Neural expression of hikaru genki protein during embryonic and larval development of Drosophila melanogaster

 Hikaru genki (HIG) is a putative secreted protein of Drosophila that belongs to immunoglobulin and complement-binding protein superfamilies. Previous studies reported that, during pupal and adult stages, HIG protein is synthesized in subsets of neurons and appears to be secreted to the synaptic clefts of neuron-neuron synapses in the central nervous system (CNS). Here we report the analyses of distribution patterns of HIG protein at embryonic and larval stages. In embryos, HIG was mainly observed in subsets of neurons of the CNS that include pCC interneurons and RP5 motorneurons. At third instar larval stage, this protein was detected in a limited number of cells in the brain and ventral nerve cord. Among them are the motorneurons that extend their axons to make neuromuscular junctions on body wall muscle 8. Immunoelectron microscopy showed that these axonal processes as well as the neuromuscular terminals contain numerous vesicles with HIG staining, suggesting that HIG is in a pathway of secretion at this stage. Some neurosecretory cells were also found to express this protein. These data suggest that HIG functions in the nervous system through most developmental stages and may serve as a secreted signalling molecule to modulate the property of synapses or the physiology of the postsynaptic cells. Received: 28 May 1998 / Accepted: 4 August 1998     

Differential expression of glucose transporter isoforms during embryonic stem cell differentiation

In mouse blastocysts six facilitative glucose transporter isoforms (GLUT)1-4, 8 and 9 are expressed. We have used the mouse embryonic stem (ES) cell line D3 and spontaneously differentiating embryoid bodies (EB) to investigate GLUT expression and the influence of glucose during differentiation of early embryonic cells. Both ES cells and EBs (2d-20d) expressed GLUT1, 3, and 8, whereas the isoforms 2 and 4 were detectable exclusively in EBs. Differentiation-associated expression of GLUT was analyzed by double staining with stage-specific embryonic antigen (SSEA-1), cytokeratins (CK18, 19), nestin, and desmin. Similar to trophoblast cells in mouse blastocysts the outer cell layer of endoderm-like cells showed a high GLUT3 expression in early EBs. In 20-day-old EBs no GLUT3 protein and only minor GLUT3 mRNA amounts could be detected. A minimal glucose concentration of 5 mM applied during 2 and 8 days of EB culture resulted in up-regulated GLUT4, Oct-4 and SSEA-1 levels and a delay in EB differentiation. We conclude that GLUT expression depends on cellular differentiation and that the expression is modulated by glucose concentration. The developmental and glucose-dependent regulation of GLUT strongly suggests a functional role of glucose and glucose transporters in ES cell differentiation and embryonic development.     

Ultrastructural differentiation during embryonic Drosophila myogenesis in vitro

Summary Cultures of embryonicDrosophila melanogaster cells were examined by electron microscopy and events in myogenesis were recorded. Thick and thin myofilaments, T-tubules and sarcoplasmic reticulum all appeared at about the same time, 10.5 hr. This was about 5 hr after the final division of myoblasts and about the time that muscle cells were elongating, aligning and fusing. Sarcoplasm typical of insect muscle was detected by 18.5 hr, as were myotendonal and tendocuticular junctions. Two populations of myocytes were detected, the cytoplasm of one more electron-dense than the other. The only previous report of myofibrilogenesis in invertebrate embryos had described novel mechanisms. InDrosophila embryonic material, however, the sequence of myofibrilogenesis resembled that in post-embryonic insect or vertebrate material. Mrs. Pilar Toribio-Fiorio provided excellent technical assistance, and Patricia Minter, the secretarial expertise. This investigation was supported, in part, by NIH Grant NS9330 and the James Douglas Research Fund to Robert L. Seecof and NIH Grant No. 1 RO1 CA17223-01 to Raymond L. Teplitz.     

Dynamic expression of alternate splice forms of D-cbl during embryogenesis

The Cbl family of proteins act as E3 ubiquitin-protein ligases and have been associated with the down regulation of a variety of receptor tyrosine kinases. Cbl proteins associate with many different cell signalling molecules suggesting that they may have functions outside of the RING finger-mediated ubiquitin ligase activity. The Drosophila melanogaster cbl gene (D-cbl) encodes two splice forms (Oncogene 19 (2000) 3299). Here we report on the differential expression of these isoforms during Drosophila embryogenesis. Both isoforms are maternally expressed but the long isoform of D-cbl is also transiently expressed in the invaginating mesoderm and later is specifically expressed in neurons of the central nervous system (CNS). Cbl protein is shown to be localised to axons of the longitudinal connectives and commissures in the central nervous system.     

Differential expression of the cellular src gene during vertebrate development

Cellular genes that are homologous to the transforming genes of certain RNA tumor viruses are suspected to play a functional role during normal developmental processes. To investigate this further, we are studying the expression of the cellular homolog of the Rous sarcoma virus transforming gene (c-src) during embryogenesis of fish, frog, and chicken by quantitative determination of the activity of the c-src encoded protein kinase (pp60^c-src). The kinase activity from embryos of fish, frog, and chicken displays the same enzymatic characteristics as the kinase from adult animals: It phosphorylates only tyrosine residues in protein substrates, and its activity is relatively insensitive to inhibition by the diadenosine nucleotide Ap4A. During the course of development, the varying kinase activity level reflects differential expression of the c-src gene product. The kinase activity is low during early development, increases dramatically during organogenesis, and decreases thereafter to the level found in adult animals. The kinase activity displays an organ specificity, with brain showing the highest activity in embryos as well as in adults. Muscle, however, shows high activities during organogenesis, but no or barely detectable activity in adult animals. Our data suggest, therefore, that the c-src gene product plays more of a role in differentiation than in proliferation processes during embryogenesis, and that it may act as a pleiotropic effector.     

Differential expression of mRNAs for PACAP and its receptors during neural differentiation of embryonic stem cells

The expressions of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and their receptors (PAC1, VPAC1 and VPAC2) were examined in the five steps of the in vitro neuronal culture model of embryonic stem (ES) cell differentiation. mRNAs for PACAP, VIP, PAC1 receptor, and VPAC2 receptor were moderately expressed in neural stem cell-enriched cultures, while VPAC1 receptor mRNA was most prominently expressed in embryoid bodies (EBs). The expression of PAC1 receptor mRNA was further upregulated after terminal differentiation into neurons. In contrast, the expressions of PAC1 receptor and PACAP mRNAs were markedly decreased after glial differentiation. These results suggest that this in vitro neuronal culture system will be a useful model for future studies on the functional role of the PACAPergic system during different stages of neuronal development.     

Regulation of larval cuticle protein gene expression in Drosophila melanogaster

Genes that encode 3rd instar larval cuticle proteins (LCP's) of Drosophila melanogaster are located in at least two chromosomal sites. The genes encoding four of the five predominant LCP's are located in a cluster at the chromosomal region 44D. They are organized in pairs that are transcribed divergently, and expressed with different timing during the third larval instar. Towards understanding the basis of gene regulation within the 44D cluster, we have analyzed genetic variants, including the 2-3 variant, which has an insertion of a copia-like transposable element, H.M.S. Beagle, within the 44D cluster. The Beagle element appears to inactivate the LCP-3 gene by inserting into its TATA box, but also may cause the precocious expression of two other LCP genes, LCP-1 and LCP-f2, in the cluster. The long terminal repeat (LTR) of the Beagle element apparently contains a sequence, perhaps an enhancer-like element, which causes altered expression of these genes. We have also investigated the cis-regulatory elements involved in expression of the LCP-2 gene in wild-type larvae. We have identified two upstream regions that may contain separate cis-regulatory elements. The region between -252 bp and -515 bp may be essential for any expression of LCP-2. Additionally, the region between -515 bp and -795 bp appears to be required for the normal level of expression of the LCP-2 gene.     

The embryonic development of larval muscles in Drosophila

Each of the abdominal hemisegments A2-A7 in the Drosophila larva has a stereotyped pattern of 30 muscles. The pattern is complete by 13 h after egg laying, but the development of individual muscles has begun with the definition of precursors at least by the onset of germ band shortening, some 5.5 h earlier. The earliest signs of muscle differentiation are cell fusions, which occur in the ventralmost mesoderm overlying the CNS and at stereotyped positions in the rest of the mesoderm as the germ band shortens. At the end of shortening, the pattern of muscle precursors produced by these fusions is complete. Precursors filled with dye reveal extensive fine processes probably involved initially in cell fusion and, subsequently, in navigation over the epidermis to form attachment points. The muscle pattern is formed before innervation and without cell death. Thus, neither of these processes is involved in determining the distribution of precursors. Evidence is presented for the view that the development of the larval muscle pattern in Drosophila depends on a prior segregation of founder cells at appropriate locations in the mesoderm with which other cells fuse to form the precursors.     

Primary sequence and developmental expression of a novel Drosophila melanogaster src gene.

We have sequenced a cDNA clone for the Drosophila melanogaster gene Dsrc28C, a homolog of the vertebrate gene c-src. The cDNA contains a single open reading frame encoding a protein of 66 kilodaltons which contains features highly conserved within the src family of tyrosine protein kinases. Novel structural features of the Dsrc28C protein include a basic pI and a polyglycine domain near the amino terminus. Cell-free translation of in vitro-transcribed RNA yielded a protein of the predicted size which could be immunoprecipitated by anti-v-src antisera. RNA blot hybridization revealed that the gene is expressed predominantly during embryogenesis, in imaginal disks of third-instar larvae, and in adult females. In situ hybridization showed that expression in adult females is largely confined to nurse cells and developing oocytes.     

Differential expression of two somatostatin genes during zebrafish embryonic development

We have identified the cDNAs of two new zebrafish preprosomatostatins, PPSS1 and PPSS3, in addition to the previously cloned PPSS2 (Argenton et al., 1999). PPSS1 is the orthologue of mammalian PPSSs, with a conserved C-terminal SS-14 sequence, PPSS2 is a divergent SS precursor and PPSS3 is a cortistatin-like prohormone. Using whole-mount in situ hybridisation, we have analysed the expression of PPSS1 and PPSS2 in zebrafish embryos up to 5 days post fertilisation. PPSS1 was expressed in the developing pancreas and central nervous system (CNS), whereas PPSS2 expression was exclusively pancreatic. In the CNS, PPSS1 was detected in several areas, in particular in the vagal motor nucleus and in cells that pioneer the tract of the postoptic commissure. PPSS1 was also expressed transiently in the telencephalon and spinal motor neurons. In all areas but the telencephalon PPSS1 was coexpressed with islet-1.     

Tissue distribution of PEBBLE RNA and pebble protein during Drosophila embryonic development

pebble (pbl) is required for cytokinesis during postblastoderm mitoses (Hime, G., Saint, R., 1992. Zygotic expression of the pebble locus is required for cytokinesis during the postblastoderm mitoses of Drosophila. Development 114, 165–171; Lehner, C.F., 1992. The pebble gene is required for cytokinesis in Drosophila. J. Cell Sci. 103, 1021–1030) and encodes a putative guanine nucleotide exchange factor (RhoGEF) for Rho1 GTPase (Prokopenko, S.N., Brumby, A., O'Keefe, L., Prior, L., He, Y., Saint, R., Bellen, H.J., 1999. A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev. 13, 2301–2314). Mutations in pbl result in the absence of a contractile ring leading to a failure of cytokinesis and formation of polyploid multinucleate cells. Analysis of the subcellular distribution of PBL demonstrated that during mitosis, PBL accumulates at the cleavage furrow at the anaphase to telophase transition when assembly of a contractile ring is initiated (Prokopenko, S.N., Brumby, A., O'Keefe, L., Prior, L., He, Y., Saint, R., Bellen, H.J., 1999. A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev. 13, 2301–2314). In addition, levels of PBL protein cycle during each round of cell division with the highest levels of PBL found in telophase and interphase nuclei. Here, we report the expression pattern of pbl during embryonic development. We show that PEBBLE RNA and PBL protein have a similar tissue distribution and are expressed in a highly dynamic pattern throughout embryogenesis. We show that PBL is strongly enriched in dividing nuclei in syncytial embryos and in pole cells as well as in nuclei of dividing cells in postblastoderm embryos. Our expression data correlate well with the phenotypes observed in pole cells and, particularly, with the absence of cytokinesis after cellular blastoderm formation in pbl mutants.