Dynamic developmental elaboration of N-linked glycan complexity in the Drosophila melanogaster embryo

The structural diversity of glycoprotein N-linked oligosaccharides is determined by the expression and regulation of glycosyltransferase activities and by the availability of the appropriate acceptor/donor substrates. Cells in different tissues and in different developmental stages utilize these control points to manifest unique glycan expression patterns in response to their surroundings. The activity of a Toll-like receptor, called Tollo/Toll-8, induces a pattern of incompletely defined, but neural specific, glycan expression in the Drosophila embryo. Understanding the full extent of the changes in glycan expression that result from altered Tollo/Toll-8 signaling requires characterization of the complete N-linked glycan profile of both wild-type and mutant embryos. N-Linked glycans harvested from wild-type or mutant embryos were subjected to direct structural analysis by analytic and preparative high pressure liquid chromatography, by multidimensional mass spectrometry, and by exoglycosidase digestion, revealing a predominance of high mannose and paucimannose glycans. Di-, mono-, and nonfucosylated forms of hybrid, complex biantennary, and triantennary glycans account for 12% of the total wild-type glycan profile. Two sialylated glycans bearing N-acetylneuraminic acid were detected, the first direct demonstration of this modification in Drosophila. Glycan profiles change during normal development consistent with increasing alpha-mannosidase II and core fucosyl-transferase enzyme activities, and with decreasing activity of the Fused lobes processing hexosaminidase. In tollo/toll-8 mutants, a dramatic, expected loss of difucosylated glycans is accompanied by unexpected decreases in monofucosylated and nonfucosylated hybrid glycans and increases in some nonfucosylated paucimannose and biantennary glycans. Therefore, tollo/toll-8 signaling influences flux through several processing steps that affect the maturation of N-linked glycans.     

dXNP, a Drosophila homolog of XNP/ATRX, induces apoptosis via Jun-N-terminal kinase activation

XNP/ATRX, a causative gene of X-linked alpha-thalassemia/mental retardation syndrome, encodes an SNF2 family ATPase/helicase protein. To better understand the role of XNP/ATRX in development, we isolated and characterized a Drosophila XNP/ATRX homolog, dXNP, which contains highly conserved SNF2 and helicase domains. Ectopically expressed dXNP induced strong apoptosis in the developing eye and wing, but did not affect cell cycle progression or the expression of wingless and engrailed, essential regulators of development. The dXNP-induced apoptosis was strongly suppressed by DJNKK/hemipterous mutation, and dXNP increased JNK activity. Taken together, these results suggest that dXNP regulates apoptosis via JNK activation.     

Cloning and characterization of Dfak56, a homolog of focal adhesion kinase, in Drosophila melanogaster.

The focal adhesion kinase (FAK) protein-tyrosine kinase plays important roles in cell adhesion in vertebrates. Using polymerase chain reaction-based cloning strategy, we cloned a Drosophila gene that is homologous to the vertebrate FAK family of protein-tyrosine kinases. We designated this gene Dfak56 and characterized its gene product. The overall protein structure and deduced amino acid sequence of Dfak56 show significant similarity to those of FAK and PYK2. Dfak56 has in vitro autophosphorylation activity at tyrosine residues. Expression of the Dfak56 mRNA and the protein was observed in the central nervous system and the muscle-epidermis attachment site in the embryo, where Drosophila position-specific integrins are localized. The results suggest that like FAK in vertebrates, Dfak56 functions downstream of integrins. Dfak56 was tyrosine-phosphorylated upon integrin-dependent attachment of the cell to the extracellular matrix. We conclude that the Dfak56 tyrosine kinase is involved in integrin-mediated cell adhesion signaling and thus is a functional homolog of vertebrate FAK.     

Identification and expression pattern of Bmlark, a homolog of the Drosophila gene lark in Bombyx mori.

Two Bombyx mori isoforms of the gene lark, which is shown to play an important role in Drosophila circadian rhythms, were identified and named Bmlark-PA and Bmlark-PB, respectively. Bmlark-PA consists of 5 exons and encodes a protein of 343 amino acid residues which contains 3 functional domains: two RRM (RNA recognization motif) domains and an RTZF (retroviral-type zinc finger) and shares 72% identity with the Drosophila gene lark at the amino acid level. Bmlark-PB lacks the sequence between 118 and 791 nt of Bmlark-PA and codes for a protein of 68 amino acid residues, which contains no distinct functional domains. Alignments of the cDNAs of Bmlark to the genomic draft sequence of B. mori showed that the gene Bmlark had a single copy in the genome, suggesting that an alternative splicing mechanism occurs in the gene Bmlark. RT-PCR analysis indicated that Bmlark-PA was expressed only in late pupae and adult but Bmlark-PB was broadly expressed in many tissues and throughout the developmental stages from embryo to adult.     

DNA sequence, structure, and tyrosine kinase activity of the Drosophila melanogaster Abelson proto-oncogene homolog.

We report our molecular characterization of the Drosophila melanogaster Abelson gene (abl), a gene in which recessive loss-of-function mutations result in lethality at the pupal stage of development. This essential gene consists of 10 exons extending over 26 kilobase pairs of genomic DNA. The DNA sequence encodes a protein of 1,520 amino acids with strong sequence similarity to the human c-abl proto-oncogene beginning in the type lb 5' exon and extending through the region essential for tyrosine kinase activity. When the tyrosine kinase homologous region was expressed in Escherichia coli, phosphorylation of proteins on tyrosine residues was observed with an antiphosphotyrosine antibody. These results show that the abl gene is highly conserved through evolution and encodes a functional tyrosine protein kinase required for Drosophila development.     

Differential expression of the adhesion molecule Echinoid drives epithelial morphogenesis in Drosophila

Epithelial morphogenesis requires cell movements and cell shape changes coordinated by modulation of the actin cytoskeleton. We identify a role for Echinoid (Ed), an immunoglobulin domain-containing cell-adhesion molecule, in the generation of a contractile actomyosin cable required for epithelial morphogenesis in both the Drosophila ovarian follicular epithelium and embryo. Analysis of ed mutant follicle cell clones indicates that the juxtaposition of wild-type and ed mutant cells is sufficient to trigger actomyosin cable formation. Moreover, in wild-type ovaries and embryos, specific epithelial domains lack detectable Ed, thus creating endogenous interfaces between cells with and without Ed; these interfaces display the same contractile characteristics as the ectopic Ed expression borders generated by ed mutant clones. In the ovary, such an interface lies between the two cell types of the dorsal appendage primordia. In the embryo, Ed is absent from the amnioserosa during dorsal closure, generating an Ed expression border with the lateral epidermis that coincides with the actomyosin cable present at this interface. In both cases, ed mutant epithelia exhibit loss of this contractile structure and subsequent defects in morphogenesis. We propose that local modulation of the cytoskeleton at Ed expression borders may represent a general mechanism for promoting epithelial morphogenesis.     

fumble encodes a pantothenate kinase homolog required for proper mitosis and meiosis in Drosophila melanogaster

A number of fundamental processes comprise the cell division cycle, including spindle formation, chromosome segregation, and cytokinesis. Our current understanding of these processes has benefited from the isolation and analysis of mutants, with the meiotic divisions in the male germline of Drosophila being particularly well suited to the identification of the required genes. We show here that the fumble (fbl) gene is required for cell division in Drosophila. We find that dividing cells in fbl-deficient testes exhibit abnormalities in bipolar spindle organization, chromosome segregation, and contractile ring formation. Cytological analysis of larval neuroblasts from null mutants reveals a reduced mitotic index and the presence of polyploid cells. Molecular analysis demonstrates that fbl encodes three protein isoforms, all of which contain a domain with high similarity to the pantothenate kinases of A. nidulans and mouse. The largest Fumble isoform is dispersed in the cytoplasm during interphase, concentrates around the spindle at metaphase, and localizes to the spindle midbody at telophase. During early embryonic development, the protein localizes to areas of membrane deposition and/or rearrangement, such as the metaphase and cellularization furrows. Given the role of pantothenate kinase in production of Coenzyme A and in phospholipid biosynthesis, this pattern of localization is suggestive of a role for fbl in membrane synthesis. We propose that abnormalities in synthesis and redistribution of membranous structures during the cell division cycle underlie the cell division defects in fbl mutant cells.     

Identification of mitogen-responsive ribosomal protein S6 kinase pp90rsk, a homolog of Xenopus S6 kinase II, in chicken embryo fibroblasts.

Antiserum raised against recombinant Xenopus ribosomal protein S6 kinase (rsk) was used to identify a 90,000-Mr ribosomal S6 kinase, pp90rsk, in chicken embryo fibroblasts. Adding serum to cells stimulated the phosphorylation of pp90rsk on serine and threonine residues and increased the activity of S6 kinase measured in immune complex assays. Xenopus S6 kinase II and chicken embryo fibroblast pp90rsk had nearly identical phosphopeptide maps.     

Computational models for neurogenic gene expression in the Drosophila embryo

The early Drosophila embryo is emerging as a premiere model system for the computational analysis of gene regulation in development because most of the genes, and many of the associated regulatory DNAs, that control segmentation and gastrulation are known. The comprehensive elucidation of Drosophila gene networks provides an unprecedented opportunity to apply quantitative models to metazoan enhancers that govern complex patterns of gene expression during development. Models based on the fractional occupancy of defined DNA binding sites have been used to describe the regulation of the lac operon in E. coli and the lysis/lysogeny switch of phage lambda. Here, we apply similar models to enhancers regulated by the Dorsal gradient in the ventral neurogenic ectoderm (vNE) of the early Drosophila embryo. Quantitative models based on the fractional occupancy of Dorsal, Twist, and Snail binding sites raise the possibility that cooperative interactions among these regulatory proteins mediate subtle differences in the vNE expression patterns. Variations in cooperativity may be attributed to differences in the detailed linkage of Dorsal, Twist, and Snail binding sites in vNE enhancers. We propose that binding site occupancy is the key rate-limiting step for establishing localized patterns of gene expression in the early Drosophila embryo.     

Differential requirements of the fused kinase for hedgehog signalling in the Drosophila embryo.

The two signalling proteins, Wingless and Hedgehog, play fundamental roles in patterning cells within each metamere of the Drosophila embryo. Within the ventral ectoderm, Hedgehog signals both to the anterior and posterior directions: anterior flanking cells express the wingless and patched Hedgehog target genes whereas posterior flanking cells express only patched. Furthermore, Hedgehog acts as a morphogen to pattern the dorsal cuticle, on the posterior side of cells where it is produced. Thus responsive embryonic cells appear to react according to their position relative to the Hedgehog source. The molecular basis of these differences is still largely unknown. In this paper we show that one component of the Hedgehog pathway, the Fused kinase accumulates preferentially in cells that could respond to Hedgehog but that Fused concentration is not a limiting step in the Hedgehog signalling. We present direct evidence that Fused is required autonomously in anterior cells neighbouring Hedgehog in order to maintain patched and wingless expression while Wingless is in turn maintaining engrailed and hedgehog expression. By expressing different components of the Hedgehog pathway only in anterior, wingless-expressing cells we could show that the Hedgehog signalling components Smoothened and Cubitus interruptus are required in cells posterior to Hedgehog domain to maintain patched expression whereas Fused is not necessary in these cells. This result suggests that Hedgehog responsive ventral cells in embryos can be divided into two distinct types depending on their requirement for Fused activity. In addition, we show that the morphogen Hedgehog can pattern the dorsal cuticle independently of Fused. In order to account for these differences in Fused requirements, we propose the existence of position-specific modulators of the Hedgehog response.     

Sequence and expression of DmMKLP1, a homolog of the human MKLP1 kinesin-like protein from Drosophila melanogaster

 We have isolated the Drosophila gene DmMKLP1, which has a high similarity to members of the mitotic kinesin-like subfamily of kinesin proteins. DmMKLP1 has no known close relatives in the Drosophila genome and can therefore be assumed to be the ortholog of human MKLP1 and hamster CHOI kinesin-like proteins. In situ hybridization reveals a homogeneous maternal expression in the early embryo and a terminally restricted expression pattern at blastoderm stage. Later, the expression becomes increasingly restricted to the developing central nervous system, where it remains expressed at least until the end of embryogenesis. Received: 15 April 1998 / Accepted: 7 May 1998     

A regulatory code for neurogenic gene expression in the Drosophila embryo

Bioinformatics methods have identified enhancers that mediate restricted expression in the Drosophila embryo. However, only a small fraction of the predicted enhancers actually work when tested in vivo. In the present study, co-regulated neurogenic enhancers that are activated by intermediate levels of the Dorsal regulatory gradient are shown to contain several shared sequence motifs. These motifs permitted the identification of new neurogenic enhancers with high precision: five out of seven predicted enhancers direct restricted expression within ventral regions of the neurogenic ectoderm. Mutations in some of the shared motifs disrupt enhancer function, and evidence is presented that the Twist and Su(H) regulatory proteins are essential for the specification of the ventral neurogenic ectoderm prior to gastrulation. The regulatory model of neurogenic gene expression defined in this study permitted the identification of a neurogenic enhancer in the distant Anopheles genome. We discuss the prospects for deciphering regulatory codes that link primary DNA sequence information with predicted patterns of gene expression.     

Cloning, chromosomal organization and expression analysis of Neurl, the mouse homolog of Drosophila melanogaster neuralized gene

The Drosophila neuralized (neur) gene belongs to the neurogenic group of genes involved in regulating cell-cell interactions required for neural precursor development. neur mutant phenotypes include strong overcommitment to neural fates at the expense of epidermal fates. The human neuralized homolog (NEURL) has been recently determined and found to map to chromosome 10q25.1 within the region frequently deleted in malignant astrocytomas. Because of its potential importance in developmental processes, we analyzed the structure of the mouse homolog, Neurl, and its expression pattern in embryonic tissues. Neurl activity is detected from early developmental stages in several tissues and organs including neural tissues, limbs, the skeletal system, sense organs and internal organs undergoing epithelial-mesenchymal interactions. Neurl encodes a polypeptide associated with the plasma membrane but also detected in the cytoplasm. Similarly to the Drosophila gene, mammalian neuralized may code for an important regulatory factor.