C. DEPARTMENT OF DEVELOPMENTAL GENETICS
C-a. Division of Developmental Genetics - Yasushi Hiromi Group

RESEARCH ACTIVITIES

(1) Multi-dimensional axon network formation

Masaki Hiramoto and Yasushi Hiromi

--A key feature of the CNS that allows efficient information processing is its repetitive modular structure. Such structure is found in the invertebrate ventral nerve cord, the vertebrate rhombomere or the columnar structures of the cortex. An individual module contains the complement of cell types and guidance cues required to form a neural circuit within the module. To construct a neural network, however, multiple modules must be interconnected by axons exiting the module border and then re-entering the next module. Such “boundary crossing" requires axons to adopt navigation strategies that are different from the rules that shaped their trajectories within the module unit. In bilateralians a boundary crossing problem occurs at the midline, the axis of symmetry. In animals with segmentally repeated body plan, an additional boundary crossing problem resides at the border of the repeat unit, segment boundary. In contrast to the well-studied midline crossing event, segmetal boundary crossing problem has never been subjected to an analysis, either in the vertebrate or the invertebrate CNS. We showed that two evolutionary conserved axon guidance molecules, secreted ligand Netrin and a guidance receptor ROBO, which are known to play important role in axon guidance across the midline, are also used as a molecular cassette to license longitudinal axons to cross the segmental boundary, thereby connecting modular structures in the CNS. This finding demonstrates that the dimension of the multi-modular structure can be increased by using the same molecular cassette multiple times.

(2) Intrinsic sub-axonal patterning in Drosophila neurons

Takeo Katsuki, Masaki Hiramoto and Yasushi Hiromi

--During the development of the nervous system, neurons extend their axons over a long distance to their targets with the assistance of guidance cues and guidance receptors. Although a number of molecules that play instructive role in axonal navigation have been discovered, little is known about how the distributions of such guidance molecules are regulated in space and time. Immunohistochemical studies have revealed that the spatial distribution of guidance receptors in vivo is often restricted to specific segments of axons. Is such sub-axonal localization of guidance receptors generated by the intrinsic properties of neurons or by extrinsic signals? And how is the localization of transmembrane receptors maintained in the axonal membrane despite the fact that the membrane is continuous and fluid? Using a primary cell culture system of Drosophila, we demonstrated that isolated neurons possess an intrinsic property to generate sub-axonal localization of guidance molecules. FRAP analysis provided evidence that an underlying mechanism for the sub-axonal localization is compartmentalization of the axonal membrane by a diffusion barrier. We propose that the regulated expression of guidance receptors in vivo is based on the intrinsic sub-axonal patterning property of neurons.

(3) Identification of BP102 antigen which shows specific sub-axonal localization

Tohru Umemura, Takeo Katsuki and Yasushi Hiromi

--A striking feature of the neuron is its regionalized cellular organization. Cellular compartments such as axon and dendrite have distinct molecular signatures that enable specific physiological functions. Axons themselves have regional specifications, such as the growth cone and the distal end and the initial segment at the base, which differ in cytoskeletal organizations that influence the dynamics of the axonal membrane and the movement of molecules within. Although the axonal shaft is seemingly uniform, a number of molecules are localized to specific sub-segments of axons in vivo. Such sub-axonal localization of molecules is likely based on intrinsic patterning ability of the neuron that regionalizes the axon to sub-axonal “compartments" (see above). One of the molecules that exhibit intrinsic sub-axonal localization is the antigen of the monoclonal antibody BP102, which stains many axons in the Drosophila CNS highlighting the ladder-like axonal scaffold. In primary culture this antibody stains the middle or proximal segment of the axon, suggesting the existence of distinct sub-axonal compartments. We are trying to identify the BP102 antigen to investigate how it organizes itself to such sub-axonal compartments. We found that the antigen is a membrane protein of about 100kD, and requires glycosilation to be recognized by the antibody.

(4) Lamina-specific connection in mouse hippocampus

Fumikazu Suto, Hajime Fujisawa1 and Yasushi Hiromi (1Nagoya University)

--In many part of central nervous system, distinct populations of axons confine their terminal arbors and synapses to different subsets of laminae. These lamina-specificities can be divided into two categories, cellular and subcellular. Several molecules that are involved in lamina-specific projection at the cellular-specific level have recently been identified in the Drosophila visual system. However, little is known about the molecular mechanisms underlying the connection to a specific subcellular region, a process that requires recognition of a subcellular compartment. To understand the molecular mechanisms for the subcellular-specific connection, we are using mouse hippocampus as a model system. In the hippocampus, dentate granule cells (DGCs) project their axons (mossy fibers; MFs) mainly to the proximal-most part of apical dendrites of CA3 pyramidal cells and partly to the proximal basal dendrites in subcellular-specific manner. We found that axon guidance molecule Sema6A was localized on the dendritic field of CA3 pyramidal cells, and had repulsive activity for MFs, suggesting that Sema6A might be the signal that specify subcellular specificity of the MF projection. We thus analyzed the roles of Sema6A receptors, Plexin-A2 and Plexin-A4, on MF projection using mutant and normal explants. plexin-A4-deficient DGCs projected MFs abnormally to all parts of the apical and basal dendrites of genotypically normal CA3 pyramidal cells. In contrast, CA3 pyramidal cells that are mutant for plexin-A2 failed to receive projections from genotypically normal MFs in the apical dendritic field, and received only in the proximal basal dendrite. These results suggest that during subcellular-specific MFs projection, Plexin-A4 mediates Sema6A repulsive activity in MFs, and Plexin-A2 regulates Sema6A activity and/or distribution in the CA3 field.

(5) seven-up controls switching of transcription factors that specify temporal identities of Drosophila neuroblasts

Makoto Kanai, Masataka Okabe and Yasushi Hiromi

--Drosophila neuronal stem cell neuroblasts constantly change character upon division to produce a different type of progeny at the next division. Transcription factors Hunchback (HB), KrU3968ppel (KR), Pdm (PDM), etc. are expressed sequentially in the developmental history of each neuroblast and act as determinants of birth-order identity. How neuroblast switches its expression profile from one transcription factor to the next is poorly understood. We showed that the HB-to-KR switch is directed by the nuclear receptor Seven-up. Seven-up expression is confined to a temporally restricted subsection within the neuroblast's lineage. Loss of seven-up function causes an increase in the number of HB-positive cells within several neuroblast lineages, whereas misexpression of seven-up leads to the loss of these early-born neurons. Lineage analysis provided evidence that seven-up is required to switch off HB at the proper time. Thus, seven-up modifies the self-renewal stem cell program to allow chronological change of cell fates, thereby generating neuronal diversity.

(6) A search for Seven-up-interacting molecules

Takayuki Hondoh and Yasushi Hiromi

--Seven-up is a transcription factor containing a DNA binding domain and an evolutionarily-conserved domain that has homology to the ligand binding domains of nuclear receptors. The spatio-temporal expression pattern of Seven-up is tightly linked to the cell fate; in the developing Drosophila eye, seven-up is expressed in only four of the eight photoreceptor neurons, which are transformed to another neuronal fate in seven-up mutant. In the embryonic CNS seven-up expression is temporally restricted to a subsection of the neuroblast lineage. The loss-of function mutation causes a “temporal transformation" of the fate of the neuroblast and its progeny (see above). Although Seven-up plays instructive roles in many cell fate decisions, how this transcription factor accomplishes such cell fate choices is not understood. We have prepared antibodies against Seven-up that would enable us to probe the endogenous Seven-up molecule. Using IP and ChIP methods, we are investigating the molecular environment around Seven-up.

(7) Glial-expression of Prospero integrates inputs from multiple signaling

Yoshihiro Yuasa and Yasushi Hiromi

--Glial cells in the nervous system are morphologically and functionally diverse, performing various functions such as providing a scaffold for axonal migration, insulation of axons, and supporting neuronal survival. In Drosophila, interface glia such as longitudinal glia enwraps the longitudinal axon and, in some ways, resembles oligodendrocytes in vertebrates. Longitudinal glia also plays a key role in the formation of longitudinal axon pathway. Ten longitudinal glial cells that reside in each hemisegment are generated from a single precursor that divides while migrating towards the midline. These longitudinal glial cells likely comprise multiple glial sub-types, because some of them have specific association with distinct axon pathways. One molecule that is expressed in a subset of the longitudinal glia is a homeodomain transcription factor Prospero (PROS). PROS is expressed in six out of ten longitudinal glial cells, and is required for the generation of the longitudinal axonal scaffold and neuronal survival. In order to understand the gene regulatory cascade leading to the specification of longitudinal glial cells, we analyzed how PROS expression is regulated in the longitudinal glia. We found that the combinational expression of three transcriptional factors and Notch signaling is essential for the PROS expression in the longitudinal glia.

(8) A screening for genes involving germline stem cell establishment and niche formation in Drosophila

Miho Asaoka, Shuji Shigenobu1, Satoru Kobayashi1 and Yasushi Hiromi (1NIBB)

--Stem cells play a central role in generating and maintaining most adult tissues in higher organisms. They are defined by their ability to self-renew and to produce numerous differentiated daughter cells. Stem cells in the adult tissue reside in a special microenvironment called “niche", which are thought to produce signals that support and maintain stem cell specification and function. However, how stem cell fate and niche are initially established during tissue development is currently unknown. In Drosophila, germline stem cells are derived from a subset of primordial germ cells (PGCs). We have previously shown that only the PGCs contacting somatic cells in the anterior half of embryonic gonad will become germline stem cells3). We also found that the somatic cells in the anterior half of the embryonic gonad already differ from the posterior somatic cells and likely constitute the niche in the adult. These results suggest that somatic cells in the anterior half of the embryonic gonad play a key role in the germline stem cell fate establishment and niche formation. As the first step for understanding molecular mechanisms of germline stem cell establishment and niche formation, we are focusing on genes that are expressed in the somatic cells in the anterior half of the embryonic gonad. Through EST and microarray analyses we identified 187 genes which are expressed only in the somatic cells in the embryonic gonad. In situ hybridization analyses show that some of these genes are expressed only in the anterior somatic cells in the embryonic gonad. We plan to test the function of these candidate genes in germline stem cell establishment and niche formation, using RNAi methods.

(9) The origin of the parathyroid gland

Masataka Okabe and Anthony Graham1 (1MRC, Centre for Developmental Neurobiology, King's College London)

--It has long been held that the parathyroid glands and parathyroid hormone evolved with the emergence of the tetrapods, reflecting a need for new controls on calcium homeostasis in terrestrial, rather than aquatic, environments. Developmentally, the parathyroid gland is derived from the pharyngeal pouch endoderm, and studies in mice have shown that its formation is under the control of a key regulatory gene, Gcm-2. We have used a phylogenetic analysis of Gcm-2 to probe the evolutionary origins of the parathyroid gland. We show that in chicks, as in mice, Gcm-2 is expressed in the pharyngeal pouches and the forming parathyroid gland. We find that Gcm-2 is present not only in tetrapods but also in teleosts and chondrichthyans, and that in these species, Gcm-2 is expressed within the pharyngeal pouches and internal gill buds that derive from them in zebrafish (Danio rerio), a teleost, and dogfish (Scyliorhinus canicula), a chondrichthyan. We further demonstrate that Gcm-2 is required for the formation of the internal gill buds in zebrafish. We also have identified parathyroid hormone 1/2-encoding genes in fish and show that these genes are expressed by the gills. We further show that the gills express the calcium-sensing receptor, which is used in tetrapods to monitor serum calcium levels. These results indicate that the tetrapod parathyroid gland and the gills of fish are evolutionarily related structures, and that the parathyroid likely came into being as a result of the transformation of the gills during tetrapod evolution4).

PUBLICATIONS

Papers
1. Niwa, N., Hiromi, Y. and Okabe, M. (2004). A conserved developmental program for sensory organ formation in Drosophila melanogaster. Nat. Genet. 36, 293-297.
2. Jindra, M., Gaziova, I., Uhlirova, M., Okabe, M., Hiromi, Y. and Hirose, S. (2004). Coactivator MBF1 preserves the redox-dependent AP-1 activity during oxidative stress in Drosophila. EMBO J. 23, 3538-3547.
3. Asaoka, M. and Lin, H. (2004). Germline stem cells in the Drosophila ovary descend from pole cells in the anterior region of the embryonic gonad. Development 131, 5079-5089.
4. Okabe, M. and Graham, A. (2004). The origin of the parathyroid gland. Proc. Nat. Acad. Sci. USA 101, 17716-17719.
5. 岡部正隆（2004）色覚バリアフリー.東京都眼科医会報187, 3-9.

ORAL PRESENTATIONS

1. Hiromi, Y.: Constructing an organ through intracellular patterning. Princeton University, Dept. Molecular Biology, Princeton, March, 2004.
2. Hiromi, Y.: Constructing an organ through intracellular patterning. RIKEN CDB, Kobe, April, 2004.
3. Hiromi, Y.: Intra-axonal patterning. The 14th International Workshop on the Molecular and Developmental Biology of Drosophila. Crete, June, 2004.
4. Okabe, M. and Graham, A.: The origin of the parathyroid. The 37th Annual Meeting of the Japanese Society of Developmental Biologists, Nagoya, June, 2004.
5. Hiromi, Y.: Intra-axonal patterning: its mechanism and implications. Neuro 2004, Osaka, September, 2004.
6. Suto, F., Tsuboi, M., Mizuno, H., Sanbo, M., Yagi, T., Mitchell, K., Chedotal, A., Hiromi, Y., and Fujisawa, H.: Plexin/Semaphorin signals regulate laminated mossy fiber projections in the hippocampus. Cold Spring Harbor Laboratory Meeting ﾔAxon Guidance & Neural Plasticityﾕ, Cold Spring Harbor, September, 2004.
7. Hiromi, Y.: Intra-axonal patterning: its mechanism and implications. The 8th Membrane Research Forum, Nagoya, November, 2004.
8. Asaoka, M.: The establishment of the stem cell fate in the Drosophila germline. The 4th International Symposium on the Molecular and Cell Biology of Egg- and Sperm- Coats, Shima, November, 2004.
9. 岡部正隆:副甲状腺の起源,日本動物学会第75回大会,神戸,2004年9月.
10. 広海健:軸索内パターニング:その機構と意義,「生物の発生・分化・再生」シンポジウム,東京,2004年11月.
11. 広海健:出所後,高飛びする方法教えます.総研大生命科学研究科合同セミナー,つま恋,2004年11月.
12. 広海健:The Science of Scientist Recruitment.第27回日本分子生物学会年会男女共同参画シンポジウム『女性研究者がPI（研究グループのリーダー）になるには?―「ガラスの天井」はどこにあるのか―』,神戸,2004年12月.
13. 平本正輝:特定領域研究「植物の軸と情報」主催シンポジウム『発生生物学のフロンティア』,京都,2004年12月.

POSTER PRESENTATIONS

1. Asaoka, M., Hiromi, Y. and Lin, H.: The establishment of the stem cell fate in the Drosophila female germline. Keystone Symposia "Germ Cells", Keystone, January, 2004.
2. Yuasa, Y. and Hiromi, Y.: Combinational expression of three transcriptional factors is essential for the PROS expression in the longitudinal glia. 45th Annual Drosophila Research Conference, Washington, DC, March, 2004.
3. Okabe, M. and Graham, A.: The origin of the parathyroid. 7th International Congress of Vertebrate Morphology, Miami, July, 2004.
4. Hiramoto, M. and Hiromi, Y.: Netrin distribution control by attractive and repulsive receptors. 4th Axon guidance & Neural plasticity meeting, Cold Spring Harbor, September, 2004.
5. Yuasa, Y. and Hiromi, Y.: Specification of longitudinal glia. The 10th European Symposium on Drosophila Neurobiology, Neuchatel, Switzerland, September, 2004.
6. Katsuki, T., Hiramoto, M. and Hiromi, Y.: Cell-autonomous axonal patterning in Drosophila neurons: compartment boundaries regulate the localization of Robo receptors. Axon Guidance & Neural Plasticity, Cold Spring Harbor, September, 2004.
7. Asaoka, M., Hiromi, Y. and Lin, H.: Lineage analysis of stem cell fate in Drosophila germline. Cold Spring Harbor Laboratory meeting "Germ Cells", Cold Spring Harbor, October, 2004.
8. Katsuki, T., Hiramoto, M. and Hiromi, Y.: An intrinsic ability of neurons to compartmentalize their axons. The 8th Membrane Research Forum, Nagoya, November, 2004.
9. Asaoka, M., Hiromi, Y. and Lin, H.: Lineage analysis of stem cell fate in Drosophila germline. The 17th Naito Conference "Molecular Basis for Maintenance and Differentiation of Stem Cells [I]". Miura-gun, November, 2004.
10. 清水裕,岡部正隆:神経系の進化的起源に関する仰天仮説,日本動物学会第75回大会,神戸,2004年9月.
11. 勝木健雄,平本正輝,広海健:細胞自律的な軸索内のコンパートメント化と分子の軸索内局在.「生物の発生・分化・再生」第3回公開シンポジウム,東京,2004年11月.
12. Kanai, M., Okabe, M. and Hiromi, Y.: Function of seven-up in Drosophila CNS development.第27回日本分子生物学会年会,神戸,2004年12月.
13. 浅岡美穂,北館祐,重信秀治,小林悟,広海健:ショウジョウバエにおける生殖幹細胞ニッチの形成機構,第27回日本分子生物学会年会,神戸,2004年12月.

EDUCATION
1. Dr. Y. Hiromi gave a lecture course at Tokyo University of Agriculture and Technology. December, 2004 (in Japanese).

SOCIAL CONTRIBUTIONS AND OTHERS

1. Dr. Y. Hiromi served as an editor for Development, Growth & Differentiation.
2. Dr. Y. Hiromi served as a member of the council of The Genetics Society of Japan.
3. Dr. M. Okabe developed “Barrier-free presentation that is friendly to colorblind people", and enhanced its public awareness through web pages:
--1)岡部正隆,伊藤啓 http://www.nig.ac.jp/color/
--2)Okabe, M. and Ito, K. http://jfly.iam.u-tokyo.ac.jp/color/
4. Dr. M. Asaoka contributed in making a DVD/Video「幹細胞とニッチ―見えてきた造血幹細胞のすみか―」（制作:桜映画社,企画:中外製薬㈱,監修:平嶋邦猛,浅野茂隆,中畑龍俊,中内啓光）.