Published papers

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger that releases Ca2+ from its intracellular stores. The InsP3 receptor has been purified and its cDNA has been cloned. We have found that the InsP3 receptor is identical to P400 protein, first identified as a protein enriched in cerebellar Purkinje cells. We have generated an L-fibroblast cell transfectant that produces cDNA-derived InsP3 receptors. The protein displays high affinity and specificity for InsP3. InsP3 induces greater Ca2+ release from membrane vesicles from transfected cells than from those from control L-fibroblasts. After incorporation of the purified InsP3 receptor into lipid bilayers InsP3-induced Ca2+ currents were demonstrated. These results suggest that the InsP3 receptor is involved in physiological Ca2+ release. Immunogold labelling using monoclonal antibodies against the receptor showed that it is highly concentrated on the smooth-surfaced endoplasmic reticulum and slightly on the outer nuclear membrane and rough endoplasmic reticulum; no labelling of Golgi apparatus, mitochondria and plasmalemma was seen. Cross-linking experiments showed that the receptor forms a homotetramer. The approximately 650 N-terminal amino acids are highly conserved between mouse and Drosophila, and this region contains the critical sequences for InsP3 binding. We have investigated the heterogeneity of the InsP3 receptor using the polymerase chain reaction and have found novel subtypes of the mouse InsP3 receptor that are expressed in a tissue-specific and developmentally specific manner.

The Drosophila strawberry (sty) locus was isolated by P-element insertion mutagenesis in a screen for mutations affecting eye development. Analysis of the mutant phenotype and the putative expression pattern of the sty gene suggested that it has multiple functions. Mutations in the sty gene lead to irregular spacing of ommatidia, an increase in the number of photoreceptor cells, as well as abnormal axonal projections to the lamina and disrupted structure of the optic lobes in the adult fly. The sty mutation also causes abnormal head involution, a change in a number of sensilla in the antennomaxillary complex in the embryonic stage and abnormal morphogenesis of the maxillary palp and wings in later stages. We examined the presumptive expression of the sty gene during development by histochemical staining for lacZ expression from enhancer trap elements inserted within the sty gene. During embryogenesis, expression of lacZ showed a segmental pattern in the ectoderm and in the nervous system. In the eye imaginal discs, lacZ was expressed in photoreceptor cells beginning a few rows posterior to the morphogenetic furrow. The lacZ was also expressed in the wing disc. In the adult, lacZ was expressed in the retina and lamina. We cloned the sty gene by P-element tagging and found that it encodes a putative secreted protein containing a cysteine-rich region similar to the epidermal growth factor (EGF) repeat. On the basis of the loss of function phenotype, the expression pattern and the predicted structure of its product, we propose that sty encodes a diffusible protein acting as a signal involved in lateral inhibition within the developing nervous system and also as a factor involved either directly or indirectly in axonal guidance and optic lobe development.

To study the role of the IP3 receptor (IP3R) upon egg activation, cDNA clones encoding IP3R expressed in the Xenopus oocytes were isolated. By analyses of the primary structure and functional expression of the cDNA, Xenopus IP3R (XIP3R) was shown to have an IP3-binding domain and a putative Ca2+ channel region. Immunocytochemical studies revealed polarized distribution of XIP3R in the cytoplasm of the animal hemisphere in a well-organized endoplasmic reticulum-like structure and intensive localization in the perinuclear region of stage VI immature oocytes. In ovulated unfertilized eggs, XIP3R was densely enriched in the cortical region of both hemispheres in addition to its polarized localization. After fertilization, XIP3R showed a drastic change in its distribution in the cortical region. These results imply the predominant role of the XIP3R in both the formation and propagation of Ca2+ waves at fertilization. © 1993.

We have isolated and characterized a new guanylyl cyclase gene (dgc1) in Drosophila. The deduced amino acid sequence (683 amino acids) most closely resembled the mammalian soluble-type guanylyl cyclase α subunit. The cyclase catalytic domain was highly conserved between the mammalian and Drosophila guanylyl cyclases. The dgc1 mRNA was detected in wild-type heads but not in bodies, and its level was reduced in the mutant eyes absent (eya), indicating that dgc1 is preferentially expressed in the CNS and in the eye. The enriched distribution in the eye suggests that dgc1 may have a role in phototransduction.

The Drosophila argos gene, which encodes a secreted protein with an EGF motif, is involved in several developmental processes regulating cell-cell interactions such as eye morphogenesis. Loss-of-function mutations in the argos gene cause an increase in the number of photoreceptor cells and cone cells, impaired retinal projections to the optic lobe, and the formation of extra veins. We show here that ubiquitously expressed argos product restored all these loss-of-function phenotypes. Overexpression of argos in the wild- type background resulted in the reduced number of photoreceptor cells, cone cells, and pigment cells, which are phenotypes opposite to those of the loss- of-function mutants. The argos gene is expressed in developing wing veins. Ubiquitous argos expression caused loss of veins in a dose-dependent manner. This phenotype was enhanced by the loss-of-function rhomboid mutation, implying the possibility that argos and rhomboid play key roles in a common pathway for normal wing vein formation. We propose that argos acts as an inhibitory signal for cellular differentiation in the developing eye and wing.

Myelin proteolipid protein (PLP), the major myelin protein in the CNS, has been thought to function in myelin assembly. Thus, mutations within the gene coding for PLP (Plp) cause hypomyelination, such as the jimpy phenotype in mice and Pelizaeus-Merzbacher disease in humans. However, these mutants often exhibit premature death of oligodendrocytes, which form CNS myelin. To elucidate the functional roles of Pip gene products in the maturation and/or survival of oligodendrocytes, we produced transgenic mice overexpressing the Plp gene by introducing extra wild-type mouse Pip genes. Surprisingly, transgenic mice bearing 4 more Plp genes exhibited dysmyelination in the CNS, whereas those with 2 more Plp genes showed normal myelination at an early age (3 weeks after birth), but later developed demyelination. Overexpression of the Plp gene resulted in arrested maturation of oligodendrocytes, and the severity of arrest was dependent on the extent of overexpression. Overexpression also led to oligodendrocyte cell death, apparently caused by abnormal swelling of the Golgi apparatus. Thus, tight regulation of Plpgene expression is necessary for normal oligodendrocyte differentiation and survival, and its overexpression can be the cause of both dys- and demyelination. © 1994.

A family of neural RNA-binding proteins has recently been described in both vertebrates and invertebrates. We have identified a new member of this family, the Drosophila musashi (msi) locus, which is required for development of adult external sensory organs (sensilla). In contrast with wild-type sensilla, which contain two outer support cells, the msi mutation typically results in the appearance of extra outer support cells. The msi putative RNA-binding protein is localized to the nucleus and appears to be expressed in all cells in each sensillum and predominantly in neurons during embryogenesis. We propose that the msi protein regulates sensillum development by controlling the expression of target genes at the posttranscriptional level. © 1994.

The Drosophila melanogaster (Dm) decapentaplegic (dpp) gene product plays an essential role during several stages of Dm development. The DPP protein is a member of the transforming growth factor-β (TGF-β) superfamily and an orthologue of mammalian bone morphogenetic proteins (BMP-2 and -4). Recently, a cDNA clone encoding the mouse Ser/Thr kinase receptor specific for BMP-2/-4 (mTFRll) was isolated. Here, we describe the deduced primary structure, the cytogenetic position and expression pattern of the Dm homologue of mTFR11 (DTFR), a putative DPP receptor. The cytogenetic position of the Dm dtfr gene was mapped to 25D. DTFR has striking homology to mTFR11, especially in the cytoplasmic domain (approx. 63%), including a Ser+Gly-rich box that is characteristic of type-I receptors for the TGF-β superfamily. Although the amino acid (aa) sequence of the extracellular domain is less conserved than that of the cytoplasmic domain, the extracellular domains of these two molecules were more homologous (approx. 27%) to each other than any other receptors for the TGF-β superfamily. The spacing of Cys residues in the extracellular domain, which is considered crucial to ligand specificity, is highly conserved in these two receptors. During Dm embryonic development, its expression pattern changes in a dynamic fashion with high levels of expression in mesoderm and midgut, with some relation to dpp mutant phenotypes. © 1994.

We report the identification of a Drosophila locus, reversed polarity (repo). Weak repo alleles were viable but affected glia in the optic lobe, resulting in a reversal in polarity of the electrophysiological response to light in the adult. Strong repo alleles caused defects in embryonic glia and resulted in embryonic lethality. Expression of repo appeared to be specific to glia throughout development. In the adult visual system, repo was expressed in laminal glia, medullar glia, and subretinal cells; in the embryo, repo was expressed in nearly all of the identified glia in the central and peripheral nervous systems except midline glia. The repo gene encoded a homeo domain protein suggesting that it might be a transcriptional regulator of genes required for glial development.

Two types of determinants appear to be responsible for the generation of neural cell diversity: non-cell-autonomous and cell-autonomous cues. 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 (also referred to as strawberry or giant lens), which regulates cell-cell interaction in the developing nervous system, needs to be mentioned. On the basis of the phenotype on loss of function of argos, its expression pattern and the predicted structure of its product (a secreted protein with a putative epidermal growth factor (EGF) motif) we propose that argos encodes a diffusible protein with pleiotropic functions that acts as a signal involved in lateral inhibition within the developing nervous system and also as a factor involved in axonal guidance. As a member of the second category, I refer to the Drosophila musashi gene that is required for lineage formation and asymmetric division of precursor cells in the developing nervous system. The musashi gene encodes a neural RNA-binding protein and is thus likely to regulate the asymmetric cell division of neural precursor cells by controlling the expression of target genes at the post-transcriptional level. We have identified its mammalian homologue, mouse-musashi-1 (m-Msi-1). In the developing central nervous system (CNS), m-Msi-1 expression was highly enriched to neural precursor cells as is the expression of nestin. Based on the results of a single cell culture experiment, m-Msi-1 expression appears to be associated with multipotent cells that are capable of self-renewal and with the generation of committed precursor cells of both neurons and glia. However, fully differentiated neuronal and glial cells lost their m-Msi-1 expression. The expression of m-Msi protein showed a complementary pattern to that of another mammalian RNA-binding protein Hu, which is localized in differentiated neurons in the CNS. Based on such differential expression patterns and its similarity to the Drosophila musashi, we propose that a combination of neural RNA-binding proteins are required for the asymmetric distribution of intrinsic determinants in the developing mammalian nervous system. The Drosophila glial-specific homeobox protein, Repo, can also be classified as a cell-autonomous cue regulating cell-fate decision during neural development. Repo expression is required for terminal differentiation and for the survival of glial cells.

Activation of the membrane-bound phospholipase D (PLD) requires cytosolic factor(s), and ADP-ribosylation factor (ARF) has been identified as a cytosolic PLD activator. In the present study, we demonstrate that calmodulin (CaM) and ARF are both involved in PLD activation in rabbit peritoneal neutrophils. The PLD activity of streptolysin O-permeabilized, cytosol- depleted rabbit neutrophils was significantly enhanced when the permeabilized cells were reconstituted with bovine brain cytosol in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPγS), whereas there was little activation of the enzyme in the absence of cytosol. The GTPγS-stimulated PLD activity in the presence of cytosol was augmented on increasing the concentration of free Ca2+. The PLD activity stimulated by GTPγS and Ca2+ in this system was inhibited by the calmodulin inhibitor W-7. These findings suggest that CaM plays a role as a cytosolic PLD activator. Moreover, highly purified CaM alone, as well as partially purified ARF alone, promoted a slight stimulation of the PLD activity in permeabilized neutrophils. Interestingly, ARF-stimulated PLD activity was augmented by CaM in the presence of GTPγS and Ca2+. This augmentation was again inhibited by W-7, as well as by the structurally unrelated CaM inhibitor trifluoperazine. These data imply that CaM stimulates the PLD activity of rabbit neutrophils in concert with ARF.

The Drosophila argos gene encodes a secreted protein with an epidermal growth factor (EGF) motif, which acts as an inhibitor of cell recruitment in the developing eye and wing. Here, we have analyzed the role of argos during optic lobe development, argos expression was observed in the optic lobes throughout the developmental stages. In argos mutants, neuropiles failed to develop normally during embryonic and larval stages, and photoreceptor axons did not project properly into the lamina. Ubiquitous expression of argos, under control of the hsp70 promoter, rescued the defects in optic lobes. We have found that glial cells failed to differentiate in the larval optic lobes of argos mutants. Correspondingly, in loss-of-function repo mutants, whose glial cells also fail to differentiate, photoreceptor axons showed the impaired projection pattern similar to the argos phenotype. These results suggest that glial cells play a role for guidance of photoreceptor axons. The loss-of-function Star mutation (Star(X155)) dominantly suppressed the defects in the argos optic lobes, suggesting that these two genes act in an antagonistic fashion during optic lobe development.

A recessive mutation, hiiragi(P1), on the second chromosome of Drosophila was obtained by P element insertion mutagenesis. Flies homozygous for hiiragi(P1) have notched wing margins. Genetic interactions between hiiragi and the genes that encode components of Notch signaling, such as Notch, Hairless, Serrate and deltex, strongly support the involvement of hiiragi in the signal transduction cascade of Notch. It has been reported that Serrate and Delta, other components of Notch signaling, share EGF-like repeats and a second conserved cysteine-rich motif, and that these components interact physically with the same region of Notch. In hiiragi(P1); Serrate(D)/+ double mutants, we observed synergistic enhancement of the notched phenotype of wing margins. In contrast, Delta(FX3) had no phenotypic effect on hiiragi(P1) in hiiragi(P1); Delta(FX3)/+ double mutants. Taken together, these results indicate that hiiragi is involved in the Notch signaling cascade induced by Serrate rather than by Delta.

Inhibitory signals of cellular differentiation from differentiating cells play an important role in regulating the number and spatial distribution of distinctive types of cells in developing tissues. Several types of inhibitory mechanisms of cellular differentiation have been identified by making full use of the developmental genetics of Drosophila compound eyes. These inhibitory mechanisms are distinct from each other in their signal transduction cascades and/or their role in the pattern formation of the developing Drosophila eye. The following events occur: firstly a diffusible protein, Scabrous (Sea), is required to confer regular spacings of the founder cells, R8 cells, or preommatidial clusters in the developing eye disc via an unknown signal transduction cascade, secondly the Notch-signalling is at least required for the single-out of the R8 cells within the pre-ommatidial cluster possibly by preventing other cells in the equivalent groups from adapting fates as R8 cells. Notch-signalling activates a simple signal cascade mediating communication between the plasma membrane and nucleus not via protein phosphorylation. In contrast, a novel diffusible ligand, Argos, was likely to be required subsequently to the selection of R8 cells. Argos was shown to inhibit the activation of a receptor tyrosine kinase, DER, and the subsequent signal transduction in the Ras/MAPK cascade (the third inhibitory mechanism). We proposed that the role of Argos is to regulate the number of differentiated cells by controlling cellular differentiation and subsequent programmed cell death. The distinct roles of these inhibitory signals in the developing Drosophila eye are discussed in detail.

Calcium signaling is known to be associated with cytokinesis; however, the detailed spatio-temporal pattern of calcium dynamics has remained unclear. We have studied changes of intracellular free calcium in cleavage- stage Xenopus embryos using fluorescent calcium indictor dyes, mainly Calcium Green-1. Cleavage formation was followed by calcium transients that localized to cleavage furrows and propagated along the furrows as calcium waves. The calcium transients at the cleavage furrows were observed at each cleavage furrow at least until blastula stage. The velocity of the calcium waves at the first cleavage furrow was ~3 μ/s, which was much slower than that associated with fertilization/egg activation. These calcium waves traveled only along the cleavage furrows and not in the direction orthogonal to the furrows. These observations imply that there exists an intracellular calcium releasing activity specifically associated with cleavage furrows. The calcium waves occurred in the absence of extracellular calcium and were inhibited in embryos injected with heparin, an inositol 1,4,5-trisphosphate (InsP3) receptor antagonist. These results suggest that InsP3 receptor mediated calcium mobilization plays an essential role in calcium wave formation at the cleavage furrows.

The Drosophila argos gene encodes a secreted protein with an EGF motif which acts as an inhibitor of cellular differentiation in multiple developmental processes. To investigate the cellular pathways regulated by Argos we screened for mutations which could modify the phenotype caused by overexpression of argos. We show that the effects of argos overexpression on the eye and wing vein development are suppressed by gain-of-function mutations of the MAPKK/D-MEK gene (Dsor1/D-mek) and the MAPK/ERK-A gene (rolled) and were enhanced by loss-of-function mutations of Star. Loss-of-function mutations in components of the Ras/MAPK signaling cascade act as dominant suppressors of the phenotype caused by the argos null mutation. A loss-of-function argos mutation enhanced the overproduction of R7 neurons caused by gain-of-function alleles of Son of sevenless and Dsor1. Conversely, overexpression of argos inhibited formation of the extra R7 cells that was caused by high-level MAPK/ERK-A activity. A phenotype of the sev; argos double mutants revealed that sev is epistatic to argos. These results provide evidence that Argos negatively regulates signal transduction events in the Ras/MAPK cascade.