Published papers

There is increasing interest in the role of RNA-binding proteins during neural development. Drosophila Musashi is one of the neural RNA-binding proteins essential for neural development and required for asymmetric cell divisions in the Drosophila adult sensory organ development. Here, a novel mammalian neural RNA-binding protein, mouse Musashi-1, was identified based on the homology to Drosophila Musashi and Xenopus NRP-1. In the developing CNS, mouse-Musashi-1 protein was highly enriched in the CNS stem cell. Single-cell culture experiments indicated that mouse Musashi-1 expression is associated with neural precursor cells that are capable of generating neurons and glia. In contrast, in fully differentiated neuronal and glial cells mouse-Musashi-1 expression is lost. This expression pattern of mouse-Musashi-1 is complementary to that of another mammalian neural RNA-binding protein, Hu (a mammalian homologue of a Drosophila neuronal RNA-binding protein Elav), that is expressed in postmitotic neurons within the CNS. In vitro studies indicated that mouse-Musashi-1 possesses binding preferences on poly(G) RNA homopolymer, whereas Hu is known to preferentially bind to short A/U-rich regions in RNA. Based on their differential expression patterns and distinct preferential target RNA sequences, we believe that the mouse-Musashi-1 and Hu proteins may play distinct roles in neurogenesis, either through sequential regulatory mechanisms or differential sorting of mRNA populations during asymmetric division of neural precursor cells.

We characterized the embryonic expression pattern and mutant phenotypes of the Drosophila gene argos, which encodes a secreted protein with an epidermal growth factor motif. The argos null mutation caused an increase in chordotonal (Ch) organs in both the thoracic and the abdominal segments, whereas overexpression of the argos gene resulted in a decrease in these organs. We showed that the argos transcripts are expressed transiently in the cells surrounding the Ch organ precursor and that the gene rhomboid (rho), which is involved in the regulation of the number of Ch organs, acts epistatically to argos in this event. Our findings suggest that argos plays a role in Ch organ precursor formation and regulates the final number of Ch organs.

The inositol 1,4,5-trisphosphate (InsP3) receptor acts as an InsP3-gated Ca2+ release channel in a variety of cell types. Type 1 InsP3 receptor (IP3R1) is the major neuronal member of the IP3R family in the central nervous system, predominantly enriched in cerebellar Purkinje cells but also concentrated in neurons in the hippocampal CA1 region, caudate-putamen, and cerebral cortex. Here we report that most IP3R1-deficient mice generated by gene targeting die in utero, and born animals have severe ataxia and tonic or tonic-clonic seizures and die by the weaning period. An electroencephalogram showed that they suffer from epilepsy, indicating that IP3R1 is essential for proper brain function. However, observation by light microscope of the haematoxylin-eosin staining of the brain and peripheral tissues of IP3R1-deficient mice showed no abnormality, and the unique electrophysiological properties of the cerebellar Purkinje cells of IP3R1 deficient mice were not severely impaired.

The inositol 1,4,5-trisphosphate (IP3) receptor is a calcium ion channel involved in the release of free Ca2+ from intracellular stores. For analysis of the role of IP3-induced Ca2+ release (IICR) on patterning of the embryonic body, monoclonal antibodies that inhibit IICR were produced. Injection of these blocking antibodies into the ventral part of early Xenopus embryos induced modest dorsal differentiation. A close correlation between IICR blocking potencies and ectopic dorsal axis induction frequency suggests that an active IP3-Ca2+ signal may participate in the modulation of ventral differentiation.

Functions of genes regulating the development of Drosophila peripheral nervous systems are summarized herein. These genes can be classified into 6 groups: <1> prepattern genes, <2> proneural genes, <3> neurogenic genes, <4> neuronal precursor genes, <5> neuronal precursor type selector genes, and <6> cell-division and lineage genes. The mechanisms described herein provide excellent paradigms in the regulation of the development of other tissues in Drosophila, as well as in other organisms, including vertebrates. The roles of two different inhibitory mechanisms, i.e. Notch-signaling and Argos, in the development of Drosophila neural precursor cells are also discussed.

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is expressed abundantly in the CNS, such as in cerebellar Purkinje cells and the hippocampus. We established a tissue-specific cell-free transcription system and studied regulatory properties of the 5' upstream region of the IP3R1 gene by use of this system. Deletion analyses of the promoter revealed several cis elements that function significantly in brain nuclear extracts. Among those elements, sequences from -396 to -295 showed the most predominant cerebellum-specific positive function. Footprint analyses demonstrated a factor-binding region from -334 to -318, termed box-I, that contained an E- box consensus sequence. Electrophoretic mobility shift assay revealed CNS- related basic helix-loop-helix proteins for the box-I. Mutational studies using the function assay end competitive electrophoretic mobility shift assays demonstrated a good correlation between the box-I-binding factors and the activated transcription. Box-I-binding factors were present abundantly in adult mouse CNS, whereas their existence was restricted in embryonic and nonneural tissues. Transient chloramphenicol acetyltransferase assay for the IP3R1 promoter revealed the requirement of box-I in Neuro2a neuroblastoma cells. In the postnatal CNS, multiple basic helix-loop-helix factors are expressed abundantly, some of which are suggested to activate IP3R1 gene expression in the mammalian CNS.

To study the role of inositol 1,4,5-trisphosphate (IP3) receptors during early embryogenesis in Xenopus, we examined the temporal-spatial localization of Xenopus IP3 receptor (XIP3R), XIP3R protein is enriched in the animal hemisphere of early cleavage stage embryos and becomes localized in the ectoderm and involuted mesoderm in gastrula stage embryos. Up to tailbud stages, expression of XIP3R is observed in the mesodermal tissues and in most subregions of the central nervous system. A quantitative analysis of endogenous IP3 mass during normal early embryogenesis revealed an increase in IP3 mass first observed at early gastrula stage of 10.5 with an enrichment in the ectoderm throughout the gastrula stages, implying a potential role during gastrulation.

Tumor necrosis factor (TNF) is thought to be one of the mediators responsible for the damage of oligodendrocytes (OLGs) in multiple sclerosis (MS). We report here the involvement of the interleukin 1β-converting enzyme (ICE)/Caenorhabditis elegans gene ced-3 (CED-3) family in TNF-mediated cell death of OLGs. The addition of TNF-α to primary cultures of OLGs that express ice and cpp32 significantly decreased the number of live OLGs in 72 h. DNA fragmentation was detected in TNF-treated OLGs at 36 h with the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. Benzyloxycarbonyl-Asp-CH 2OC(O)-2,6-dichlorobenzene, an inhibitor of the ICE/CED-3 family that shows p35-like inhibitory specificity, protected against the TNF-induced cell death of OLGs. Furthermore, acetyl-YVAD-CHO (a specific inhibitor of ICE-like proteases) as well as acetyl-DEVD-CHO (a specific inhibitor of CPP32-like proteases) enhanced the survival of OLGs treated with TNF-α, indicating that ICE- and the CPP32-mediated cell death pathways are activated in TNF-induced OLG cell death. Our results suggest that the inhibition of ICE/CED-3 proteases may be a novel approach to treat neurodegenerative diseases such as MS.

The chordotonal (Ch) organ, an internal stretch receptor located in the subepidermal layer, is one of the major sensory organs in the peripheral nervous system of Drosophila melantogaster. Although the cell lineage of the Ch organ has been well characterized in many studies, the determination machinery of Ch organ precursor cells (COPs) remains largely unresolved. Here we report that the rhomboid (rho) gene and the activity of the Drosophila EGF receptor (DER) signaling pathway are necessary to induce specifically three of the eight COPs in an embryonic abdominal hemisegment. The cell-lineage analysis of COPs using the yeast flpase (flp/FRT) method indicated that each of the eight COPs originated from an individual undifferentiated ectodermal cell. The eight COPs in each abdominal hemisegment seemed to be determined by a two-phase induction: first, five COPs are determined by the action of the proneural gene atonal and neurogenic genes. Subsequently, these five COPs start to express the rho gene, and rho activates the DER-signaling pathway in neighboring cells and induces argos expression. Three of these argos-expressing cells differentiate into the three remaining COPs and they prevent neighboring cells from becoming extra COPs.

mouse-Musashi-1 (m-Msi-1) is an RNA-binding protein, abundantly expressed in the developing mammalian central nervous system (CNS). m-Msi-1 contains two RNA recognition motifs (RRMs). In this study, we found that the N-terminal RRM of m-Msi-1 (MMA) binds strongly to poly(G) and weakly to poly(U) in a way similar to that of the full-length m-Msi-1 protein characterized previously. The C-terminal RRM of m-Msi-1 (MMB), however, does not bind to RNA. In addition, the circular dichroism (CD) spectra of the two RRMs showed that the α-helical content of MMA is significantly higher than that of MMB, indicating that some differences in the secondary structure may be responsible for the distinct RNA binding properties of MMA and MMB.

To study the development of the calcium release mechanism, we examined the temporal and spatial expression of inositol 1,4,5-trisphosphate receptor (IP3R) during the oogenesis and meiotic maturation of Xenopus laevis. Observation of a series of fixed samples by immunofluorescence microscopy revealed a relocalization of Xenopus IP3R (XIP3R)-positive structures during meiotic maturation. We visualized the endoplasmic reticulum (ER) using ER-sensitive dye, DiI, by observation under confocal laser scanning microscopy. Time-lapse visualization of the living oocytes revealed that while the ER of immature fully grown oocytes underwent relatively little movement, the ER of maturing oocytes and mature eggs moved rapidly. A possible role for the increase of ER mobility in the dynamic redistribution of XIP3R during oocyte maturation is also discussed.

Cell fate in the nervous system is determined by environmental signals and/or heredity. They can be referred to as extrinsic and intrinsic determinants, respectively. The molecular and cellular mechanisms underlying these processes are well conserved from invertebrates to vertebrates. We have identified both types of determinants through the intensive screening of P-element-induced Drosophila mutants affected in neural development. As a member of the first category, argos gene product which regulates cell-cell interaction in the developing nervous system gives a good example. As a member of the second category, we refer to Drosophila musashi gene encoding a neural RNA-binding protein. The functions of these gene products and application to the mammalian nervous system will be discussed.

There is an increasing interest in the role of RNA-binding proteins during neural development. Mouse-Musashi-1 (m-Msi-1) is a mouse neural RNA- binding protein with sequence similarity to Drosophila musashi (d-msi), which is essential for neural development. m-Msi-1 is highly enriched in neural precursor cells that are capable of generating both neurons and glia during embryonic CNS development. The present study characterized m-Msi-1-expressing cells in the postnatal and adult CNS. Postnatally, m-Msi-1 was expressed in proliferative neuronal precursors in the external granule cell layer of the cerebellum and in the anterior corner of the subventricular zone of the lateral ventricles. In gliogenesis, the persistent expression of m-Msi-1 was observed in cells of the astrocyte lineage ranging from proliferative glial precursors in the subventricular zone (SVZ), to differentiated astrocytes in the parenchyma. In addition, we showed that m-Mis-1 was still expressed in proliferating cells in the adult SVZ, which may contain neural precursor or stem cells. Another neural RNA-binding protein Hu (the mammalian homolog of Drosophila neuronal RNA-binding protein Elav) was present in postmitotic neurons throughout the development of the CNS, and its pattern of expression was compared with that of m-Msi-1. These observations imply that these two RNA-binding proteins may be involved in the development of neurons and glia by regulating gene expression at the post-transcriptional level.

Molecular genetic study of Caenorhabditis elegans revealed the cell death-executing gene ced-3. Structural and functional similarities between CED-3 and mammalian ICE family (caspase) gene products provide the evidence that mammalian caspases are common mediators in executing programmed cell death. These findings suggest that the execution mechanisms of cell death by caspase family protease may be evolutionarily conserved from worms to mammals. This report summarizes the unique properties of caspases in apoptosis.

The members of ICE/CED3 family of cysteine proteases (caspases) play important roles in the regulation of programmed cell death. Recent studies have revealed that processing and activation of CPP32 (CASP-3) are crucial for executing cell death in TNF-α- and Fas-induced apoptosis. Overexpression of Bcl-2 in HeLa cells inhibits the cell death and CASP-3 like protease activities which are induced by TNF-α or anti-Fas antibody. Preform of CASP-3 disappered after the treatment with TNF-α or anti-Fas antibody in both HeLa cells and HeLa/bcl-2 cells. These results suggest Bcl-2 prevents cell death and activation of CASP-3 but does not inhibit initial processing of CASP-3.

ced-9, a member of the bcl-2 gene family in Caenorhabditis elegans plays a central roles in preventing cell death in worms. Overexpression of human bcl-2 can partially prevent cell death in C. elegans. However, it remains to be elucidated whether ced-9 can regulate cell death when expressed in other organisms. We demonstrated that the CED-9 protein is co-localized with BCL-2 in COS cells and Drosophila Schneider's L2 (SL2) cells, suggesting that the site of CED-9 action is located to specific cytoplasmic compartments. Overexpression of ced-9 only poorly protected cells from the death induced by ced-3 in HeLa cells, but ced-9 significantly reduced the cell death induced by ced-3 in Drosophila SL2 cells. Furthermore, apoptosis of SL2 cells that was induced by a Drosophila cell-death gene, reaper, was shown to be partially prevented by ced-9, bcl-2 and bcl-xL. These results suggest that the signaling pathway that is required for the anti-apoptotic function of bcl-2 family members, including ced-9, is conserved in Drosophila cells. In addition, SL2 cells provide a unique systems for dissecting the main machinery of cell death.

We have isolated a novel Drosophila paired-like homeobox gene, DPHD-1. The homeodomain of DPHD-1 showed 85% amino-acid identity with that of the C. elegans Unc-4 protein. Whole-mount in situ hybridization of embryos and third-instar larvae revealed that the DPHD-1 mRNA is specifically localized in subsets of postmitotic neurons in the central nervous system (CNS) and in the developing epidermis with a segmentally repeated pattern. Double staining with a posterior compartment marker, an anti-Engrailed antibody, showed that DPHD-1 expressing neurons in the CNS were present in the posterior compartment, whereas DPHD-1 expression in the epidermis was restricted to the anterior compartment in each segment. This temporal and spatial expression pattern suggests that DPHD-1 may play a role in determining the distinct cell types in each segment.