Our body is composed of many thousands of cell species, whose number has not yet been clarified by our most advanced researches. Especially, brain tissue is composed of many types of neurons, which is a specialized cell type that plays major roles in neuronal transmittion, and correct formation of neuronal network is required for brain functions such as memory and thinking.

How is the brain made?

This is our main question. Solving this issue seems to be very important not only to unveil the developmental mystery but also to establish new therapeutic approach for neurodegenerative disease such as Parkinson's and Alzheimer's disease.

Neurodegenerative disease is caused by selective loss of some neuronal cell types. Regeneration therapy, a reconstruction of neuronal network by transplantation or regeneration of desired neuronal cell populations, is one of the promising therapeutic approaches for neurodegenerative disease. We are aiming at developing new technologies for controlling stem cell differentiation and isolating desired neuronal populations to realize safe and efficient regeneration therapy. Moreover, these stem cell technologies are useful for drug discovery research. Recapitulating pathology in vitro using purified stem cell-derived neuronal subtypes would facilitate understanding mechanism underlying disease initiation/progression and drug screening.

Research theme 1: Mechanism of mesencephalic dopaminergic neuron development

Mesencephalic dopaminergic neurons (mesDA) control movement and behavior, and selective loss of mesDA is thought to cause Parkinson's disease. Most characteristic feature of mesDA development is their generation from the midline of the brain primordium. Morphologically and functionally specialized organizer cells named floor plate (FP) cells develop along the ventral midline from caudal diencephalon to the spinal cord. FP cells have been thought to be non-neurogenic cells that organize patterning of the ventral neuron subtypes including mesDA neurons through secreting Shh.

We identified FP-specific cell surface marker Corin (Fig. 1A). Fate mapping experiment based on cell sorting with anti-Corin antibody revealed that mesDA are generated from mesencephalic FP cells themselves (Fig. 1B), which revised the previous theory. By contrast, FP cells in the caudal neural tube (cFP) were non-neurogenic as previously thought. Therefore, FP cells that develop in different antero-posterior locations have distinct neurogenic potential (Fig. 1C).

Next, we searched factors that determine FP subtype identity and identified transcription factor Otx2, which is an anter-posterior specifier that determine fore/midbrain identity, as a master determinant of mesencephalic identity of FP cells, which confers neurogenic potential (Fig. 1D). We also identified mesDA-selective transcription factor Lmx1a, which act as a downstream of Otx2 to control neurogenic activity in FP cells (Fig. 1E,F).

Non-neurogenic cFP cells are also non-proliferating cells. In dreher mutant mice, which carry a loss-of-function mutation in the Lmx1a locus, neurogenic activity in mesencephalic FP cells was decreased but these FP cells still maintained proliferative potential, suggesting that factors other than Lmx1a control growth potential in mesencephalic FP cells. We searched FP-selective transcription factors and identified Nato3 with unknown function (Fig. 2A). Gene knockout study revealed that Nato3 is required for efficient generation of mesDA (Fig. 1B). Importantly, medial subpopulation of mesencephalic FP cells exited the cell cycle to differentiate into cFP-like cells in the Nato3 mutants. Furthermore, we found that Nato3 represses Hes1, which is a suppressor of neuronal differentiation and is required for cFP differentiation. Therefore, Nato3 appears to confer growth potential and neurogenic activity on mesencephalic FP cells by suppressing cFP-like differentiation through repressing Hes1 (Fig. 2C).

Above observations suggest that FP cells with mesencephalic context should have neurogenic activity and generate mesDA neurons. In other words, FP identity appears to determine mesDA identity. Forced expression of proneural factor Mash1 induced neurogenesis in FP cells; however, generated neurons did not have correct mesDA identity. The fact that forced expression of Otx2 in cFP cells induced generation of correct mesDA neurons suggests that Otx2 itself or its downstream factors determine mesDA identity. Indeed, it has been reported that Lmx1a have a potency to induce mesDA generation. However, mesDA-inducing activity of Lmx1a is restricted within ventral mesencephalic progenitors.

We searched factors that cooperate to determine mesDA fate and found that FP differentiation factor Foxa2 acts as cofactor for Lmx1a to induce mesDA (Fig. 3A). We also revealed that Foxa2 restricts mesencephalic progenitors to mesDA or red nucleus (RN) fate by inducing a partial differentiation program, and only in this context, Lmx1a selects a mesDA fate by repressing RN identity (Fig. 3B). In addition, RN factor Nkx6.1 has a potency to suppress FP differentiation, and thus, Lmx1a appears to induce FP differentiation by repressing Nkx6.1 expression (Fig. 3C). These observations suggest that Lmx1a and Foxa2 cooperatively coordinate mesDA specification and differentiation, and as a consequence, mesDA neurons are generated from FP cells.

Clinical trials of cell replacement therapy for Parkinson's disease using fetal human tissues as transplantation materials have proven efficacy of this approach. However, the ethical and practical problems with usage of human materials should be solved. One direction is a development of stem cell technology using ES/iPS cells, and methods to induce mesDA neurons from the stem cells have been established. However, in all of these methods, 100% of the cells are not induced to differentiate into mesDA neurons. The transplanting of cell populations including undifferentiated ES cells or neurons other than mesDA carries potential risks of tumorigenesis or side effects. Above mentioned surface marker Corin is highly selective for mesDA progenitors, and indeed, we have succeeded to enrich mouse ES cell-derived mesDA progenitors (Fig. 4A) and to eliminate undifferentiated ES cells (Fig. 4B). A trial involving an application of this approach to human ES/iPS cells is presently ongoing.

Research theme 2: Mechanism of GABA/glutamatergic transmitter phenotype selection in mesencephalic neurons

Most neurons in the brain are excitatory glutamatergic (Glut) or inhibitory GABAergic neurons. However the mechanism of how neurons select transmitter phenotype still remains mostly unknown.

We have identified a novel transcriptional repressor Helt, which is selectively expressed in GABAergic progenitors in the mesencephalon (Fig. 5A). In Helt KO embryos, generation of GABAergic neurons was mostly abolished and instead Glut was generated from presumptive GABAergic progenitors (Fig. 5B). Consistently, forced expression of Helt inhibited Glut neurogenesis and induced GABAergic neurons (Fig. 5C). We also found that Ngn genes can determine Glut fate, and that Helt represses Ngn gene expression (Fig. 5D,E). These observations demonstrated that Helt acts as a selector gene that determines GABAergic phenotype by suppressing Glut fate (Fig. 5F). This mechanism appears to be used in most neurons in the mesencephalon. In addition, our results suggest that at least in the mesencephalic neurons, transmitter phenotype is likely to be determined independently of neuronal subtype identity.

Research theme 3: Mechanism of cell fate determination of cerebellar GABAergic neurons

The cerebellum is composed of Glut, such as granule cells, and GABAergic neurons including Purkinje cells (PC). PCs play central roles in cerebellar function, whose loss causes cerebellar dysfunction in neurodegenerative disorders, such as spino-cerebellar ataxia. Despite many efforts in studies on cerebellar developmental biology, the mechanism underlying PC specification has yet to be unmasked. One reason for this difficulty is caused by the fact that developmental origin of PC has not been precisely identified probably due to the absence of early PC precursor-selective markers.

We searched PC-selective genes and identified transcriptional cofactor Corl2. Corl2 is specifically expressed in PCs in the adult cerebellum (Fig. 6A). More importantly, nascent PC precursors emerging in the embryonic cerebellum could be marked by Corl2 expression (Fig. 6B). The localization pattern of Corl2+ precursors suggested embryonic cerebellar subregion that generates PCs (Fig. 6C).

We found that cell surface protein Neph3 is selectively expressed in a subpopulation of embryonic cerebellar progenitors (Fig. 7A). Promoter analysis revealed that Neph3 is a direct downstream target gene of Ptf1a, an essential regulator for cerebellar GABAergic development. Consistently, cell sorting experiments using anti-Neph3 antibody demonstrated that Neph3 specifically marks cerebellar GABAergic progenitors. Furthermore, we found that most of Neph3+ cells sorted from E12.5 cerebellum are progenitors generating PC (Fig. 7B).

To reveal more precise origin of PC, we searched PC progenitor-selective surface markers and identified E-cadherin. Cell sorting-based fate mapping analysis revealed that Neph3+ E-cadherin+ progenitors in E12.5 cerebellum are the PC progenitors (Fig. 7C).

These observations revealed subdomains in the developing cerebellum and provide insight into the mechanism of GABAergic subtype specification in the cerebellum. We have identified PC-selective transcription factors by gene search using sorted PC progenitors, and analyses of function of these genes in PC development is currently ongoing.

Research theme 4: Mechanism of cell fate determination of basal forebrain cholinergic neurons

Basal forebrain cholinergic neurons (BFCN) are thought to play essential roles in learning and memory. In Alzheimer's disease (AD), inactivation or loss of BFCN is known to occur from early phase. Indeed, acetylcholine esterase-inhibitor is effective in the treatment on AD. Therefore, BFCN is an important target cell type for AD therapy, and efficient method of BFCN induction from stem cells would be a useful technology for drug discovery research. However, the mechanism of BFCN development remains mostly unknown, and thus, efficient induction protocol has not yet been established. Toward this end, we have analyzed developmental origin of BFCN and roughly identified embryonic brain region as their origin. Search for BFCN-selective markers is currently ongoing.

Publications

Nishimura, M., Kakizaki, M., Ono, Y., Morimoto, K., Takeuchi, M., Inoue, Y., Imai, T. and Takai, Y. (2002). JEAP, a novel component of tight junctions in exocrine cells.
J Biol Chem 277, 5583-7.

Matsui, T., Hayashi-Kisumi, F., Kinoshita, Y., Katahira, S., Morita, K., Miyachi, Y., Ono, Y., Imai, T., Tanigawa, Y., Komiya, T. et al. (2004). Identification of novel keratinocyte-secreted peptides dermokine-alpha/-beta and a new stratified epithelium-secreted protein gene complex on human chromosome 19q13.1.
Genomics 84, 384-97.

Nakatani, T., Mizuhara, E., Minaki, Y., Sakamoto, Y. and Ono, Y. (2004). Helt, a novel basic-helix-loop-helix transcriptional repressor expressed in the developing central nervous system.
J Biol Chem 279, 16356-67.

Kiyozumi, D., Osada, A., Sugimoto, N., Weber, C. N., Ono, Y., Imai, T., Okada, A. and Sekiguchi, K. (2005). Identification of a novel cell-adhesive protein spatiotemporally expressed in the basement membrane of mouse developing hair follicle.
Exp Cell Res 306, 9-23.

Minaki, Y., Mizuhara, E., Morimoto, K., Nakatani, T., Sakamoto, Y., Inoue, Y., Satoh, K., Imai, T., Takai, Y. and Ono, Y. (2005). Migrating postmitotic neural precursor cells in the ventricular zone extend apical processes and form adherens junctions near the ventricle in the developing spinal cord.
Neurosci Res 52, 250-62.

Mizuhara, E., Nakatani, T., Minaki, Y., Sakamoto, Y. and Ono, Y. (2005). Corl1, a novel neuronal lineage-specific transcriptional corepressor for the homeodomain transcription factor Lbx1.
J Biol Chem 280, 3645-55.

Mizuhara, E., Nakatani, T., Minaki, Y., Sakamoto, Y., Ono, Y. and Takai, Y. (2005). MAGI1 recruits Dll1 to cadherin-based adherens junctions and stabilizes it on the cell surface.
J Biol Chem 280, 26499-507.

Osada, A., Kiyozumi, D., Tsutsui, K., Ono, Y., Weber, C. N., Sugimoto, N., Imai, T., Okada, A. and Sekiguchi, K. (2005). Expression of MAEG, a novel basement membrane protein, in mouse hair follicle morphogenesis.
Exp Cell Res 303, 148-59.

Nakatani, T., Minaki, Y., Kumai, M. and Ono, Y. (2007). Helt determines GABAergic over glutamatergic neuronal fate by repressing Ngn genes in the developing mesencephalon.
Development 134, 2783-93.

Ono, Y., Nakatani, T., Sakamoto, Y., Mizuhara, E., Minaki, Y., Kumai, M., Hamaguchi, A., Nishimura, M., Inoue, Y., Hayashi, H. et al. (2007). Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells.
Development 134, 3213-25.

Hayashi, H., Morizane, A., Koyanagi, M., Ono, Y., Sasai, Y., Hashimoto, N. and Takahashi, J. (2008). Meningeal cells induce dopaminergic neurons from embryonic stem cells.
Eur J Neurosci 27, 261-8.

Minaki, Y., Nakatani, T., Mizuhara, E., Inoue, T. and Ono, Y. (2008). Identification of a novel transcriptional corepressor, Corl2, as a cerebellar Purkinje cell-selective marker.
Gene Expr Patterns 8, 418-23.

Jonsson, M. E., Ono, Y., Bjorklund, A. and Thompson, L. H. (2009). Identification of transplantable dopamine neuron precursors at different stages of midbrain neurogenesis.
Exp Neurol 219, 341-54.

Mizuhara, E., Minaki, Y., Nakatani, T., Kumai, M., Inoue, T., Muguruma, K., Sasai, Y. and Ono, Y. (2010). Purkinje cells originate from cerebellar ventricular zone progenitors positive for Neph3 and E-cadherin.
Dev Biol 338, 202-14.

Nakatani, T., Kumai, M., Mizuhara, E., Minaki, Y. and Ono, Y. (2010). Lmx1a and Lmx1b cooperate with Foxa2 to coordinate the specification of dopaminergic neurons and control of floor plate cell differentiation in the developing mesencephalon.
Dev Biol 339, 101-113.

Ono, Y., Nakatani, T., Minaki, Y. and Kumai, M. (2010). The basic helix-loop-helix transcription factor Nato3 controls neurogenic activity in mesencephalic floor plate cells.
Development 137, 1897-906.

Miyata, T., Ono, Y., Okamoto, M., Masaoka, M., Sakakibara, A., Kawaguchi, A., Hashimoto, M. and Ogawa, M. (2010). Migration, early axonogenesis, and Reelin-dependent layer-forming behavior of early/posterior-born Purkinje cells in the developing mouse lateral cerebellum.
Neural Dev 5, 23.

Muguruma, K., Nishiyama, A., Ono, Y., Miyawaki, H., Mizuhara, E., Hori, S., Kakizuka, A., Obata, K., Yanagawa, Y., Hirano, T. et al. (2010). Ontogeny-recapitulating generation and tissue integration of ES cell-derived Purkinje cells.
Nat Neurosci. 13, 1171-80

Issued patents

Method of identifying purkinje cell targeting Corl2 gene
Yuichi Ono, Tomoya Nakatani
Patent No.: EP1854881B1  Date of Patent: Jun. 2, 2010
Date of application: Feb. 1, 2006

Lrp4/Corin dopamine-producing neuron precursor cell marker
Yoshimasa Sakamoto, Yuichi Ono, Toshio Imai, Yasuko Nakagawa
Patent No.: JP3996627B2  Date of Patent: Aug. 10, 2007
Date of application: Jul. 22, 2005

Marker Lmx1a specific to dopamine-producing neuron
Yuichi Ono, Yasuko Nakagawa, Tomoya Nakatani
Patent No.: JP4236270B2  Date of Patent: Dec. 26, 2008
Date of application: Nov. 26, 2004
Patent No.: EP1712638B1  Date of Patent: Jun. 10, 2009
Date of application: Nov. 26, 2004

Lrp4/Corin dopamine-producing neuron proliferation precursor cell marker
Yuichi Ono, Yasuko Nakagawa, Yoshimasa Sakamoto
Patent No.: JP4573332B2  Date of Patent: Aug. 27, 2010
Date of application: Jan. 23, 2004
Patent No.: US7807371B2  Date of Patent: Oct. 5, 2010
Date of application: Apri. 25, 2008

Gene expressed specifically in dopamine-producing neuron precursor cells after termination of division
Yasuko Minaki, Yuichi Ono, Yoshimasa Sakamoto, Eri Mizuhara, Tomoya Nakatanani, Yoshimi Takai
Patent No.: JP4118877B2  Date of Patent: May. 22, 2008
Date of application: Oct. 21, 2003
Patent No.: JP4085117B2  Date of Patent: Feb. 22, 2008
Date of application: Oct. 21, 2003

Exocrine gland tight junction-constituting protein JEAP family
Miyuki Nishimura, Mayumi Asano, Yuichi Ono, Koji Morimoto, Masakazu Takeuchi, Yoko Inoue, Toshio Imai, Yoshimi Takai
Patent No.: US7824676B2  Date of Patent: Nov. 2, 2010
Date of application: Sep. 15, 2006
Patent No.: US7129063B2  Date of Patent: Oct. 31, 2006
Date of application: Nov. 15, 2002

Mouse hair follicle placode-specific extracellular matrix protein QBRICK, cDNA cloning, and human homolog
Daiji Kiyozumi, Akiko Okada, Kiyotoshi Sekiguchi, Aki Nagata, Toshio Imai, Yuichi Ono
Patent No.: JP4117359B2  Date of Patent: May. 2, 2008
Date of application: Sep. 18, 2002

Published major applications for patent

Method for obtaining pancreatic progenitor cell using Neph3
Yuichi Ono, Nakatani Tomoya, Yasuko Nakagawa
Publication No.: WO2010/073972
Date of application: Dec. 18, 2009

Method for obtaining purkinje progenitor cell by using Neph3(65B13) and E-cadherin
Yuichi Ono, Yasuko Nakagawa, Eri Mizuhara
Publication No.: WO2010/016533
Date of application: Aug. 6, 2009

GABA neuron progenitor cell marker 65B13
Yuichi Ono, Yasuko Nakagawa, Eri Mizuhara
Publication No.: WO08/096817
Date of application: Feb. 7, 2008

Dopamine-producing neuron progenitor cell marker 187A5
Yuichi Ono, Yoshimasa Sakamoto
Publication No.: WO07/119759
Date of application: Apr. 11, 2007

Msx1/2, markers of growing progenitor cell of dopamine-producing neuron
Yuichi Ono, Yasuko Nakagawa, Tomoya Nakatani, Yoshimasa Sakamoto
Publication No.: WO07/021004
Date of application: Aug. 18, 2006

Nato3, marker of growing progenitor cell of dopamine-producing neuron
Yuichi Ono, Yasuko Nakagawa, Tomoya Nakatani, Yoshimasa Sakamoto
Publication No.: WO07/021003
Date of application: Aug. 18, 2006

Method of distinguishing spinal cord neuron type targeting Corl1 gene
Yuichi Ono, Yasuko Nakagawa, Tomoya Nakatani
Publication No.: WO06/022243
Date of application: Aug. 23, 2005