Stefan Baumgartner

Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network

The sequence of paired, a pair-rule gene required for segmentation in Drosophila, is presented. A search for genes with domains homologous to the paired gene was initiated and three homologues from a set of 12 were characterized with respect to temporal or spatial expression and sequence homologies. All four are transcribed in early development, one in the oocyte and during cleavage stages in the form of a gradient. In addition to the prd-specific his-pro repeat, some of the 12 genes contain M-repeats and two new types of homeo boxes not detectable by hybridization with the two known classes of homeo boxes. The observed linking of gene sets through combinations of homologies coding for protein domains is consistent with a general network concept of gene action.

A Drosophila Neurexin Is Required for Septate Junction and Blood-Nerve Barrier Formation and Function

Septate and tight junctions are thought to seal neighboring cells together and to function as barriers between epithelial cells. We have characterized a novel member of the neurexin family, Neurexin IV (NRX), which is localized to septate junctions (SJs) of epithelial and glial cells. NRX is a transmembrane protein with a cytoplasmic domain homologous to glycophorin C, a protein required for anchoring protein 4.1 in the red blood cell. Absence of NRX results in mislocalization of Coracle, a Drosophila protein 4.1 homolog, at SJs and causes dorsal closure defects similar to those observed in coracle mutants. nrx mutant embryos are paralyzed, and electrophysiological studies indicate that the lack of NRX in glial-glial SJs causes a breakdown of the blood-brain barrier. Electron microscopy demonstrates that nrx mutants lack the ladder-like intercellular septa characteristic of pleated SJs (pSJs). These studies identify NRX as the first transmembrane protein of SJ and demonstrate a requirement for NRX in the formation of septate-junction septa and intercellular barriers.

The 55 kd regulatory subunit of Drosophila protein phosphatase 2A is required for anaphase.

The gene encoding the Drosophila protein phosphatase 2A 55 kd regulatory subunit (PR55) is located at 85F and directs the synthesis of differentially spliced transcripts. Maternal RNAs are present at very high levels in early embryos and decline around cellularization. Zygotic transcripts are present mainly in the developing embryonic nervous system and gonads. Transcripts are uniformly distributed in third instar larval discs and testes and at lower levels in the proliferative centers of the brain. Mutations in abnormal anaphase resolution (aar) are rescued by the wild-type gene for PR55. aar mutants display intact lagging chromatids that have undergone separation from their sisters, but that remain at the position formerly occupied by the metaphase plate, as well as anaphase figures that show bridging chromatin having two centromeric regions.

Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila.

The transmembrane protein Dystroglycan is a central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin, agrin and perlecan, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Using mosaic analysis and RNAi in the model organism Drosophila melanogaster, we show that Dystroglycan is required cell-autonomously for cellular polarity in two different cell types, the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a reduced apical localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin in follicle cells, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity; and this study sets the stage for analyzing the Dystroglycan complex by using the power of Drosophila molecular genetics.

tenm, a Drosophila gene related to tenascin, is a new pair-rule gene

We describe the molecular characterization of the Drosophila gene tenm, a large transcription unit spanning > 110 kb of DNA. tenm encodes a large extracellular protein of 2515 amino acids related to the extracellular matrix molecule tenascin. The Tenm protein is found in seven stripes during the blastoderm stage, and each stripe overlaps with the even‐skipped stripes. tenm mutants show a phenotype resembling that of odd‐paired (opa), a member of the pair‐rule class of segmentation genes. Thus, Tenm is the first example of a pair‐rule gene product acting from outside the cell. While the Tenm protein is under the control of fushi tarazu and even‐skipped, but not of opa, at least two pair‐rule genes, paired (prd) and sloppy paired (slp), and all segment‐polarity genes analysed to date are under the control of tenm. Our data suggest that Tenm initiates a signal transduction cascade which acts, via or in concert with opa, on downstream targets such as prd, slp, gooseberry, engrailed and wingless, leading to an opa‐like phenotype.

Isolation of two tissue-specific Drosophila paired box genes, Pox meso and Pox neuro.

Two new paired domain genes of Drosophila, Pox meso and Pox neuro, are described. In contrast to the previously isolated paired domain genes, paired and gooseberry, which contain both a paired and a homeo‐domain (PHox genes), Pox meso and Pox neuro possess no homeodomain. Evidence suggesting that the new genes encode tissue‐specific transcriptional factors and belong to the same regulatory cascade as the other paired domain genes includes (i) tissue‐specific expression of Pox meso in the somatic mesoderm and of Pox neuro in the central and peripheral nervous system, (ii) nuclear localization of their proteins, (iii) dependence on prd activity and (iv) presence of the paired domain in genes of known regulatory activity. While no mutant phenotypes of Pox meso and Pox neuro have yet been discovered, a murine gene with a paired domain closely homologous to that of Pox meso has recently been identified with the undulated mutant. Both Pox meso and undulated are expressed in tissues derived from the somatic mesoderm. The five known Drosophila paired domains fall into three classes: (i) the prd,gsb‐class, (ii) the Pox meso, undulated‐class and (iii) the Pox neuro‐class which probably includes the paired domain of the murine gene Pax 2.

Formation of the bicoid morphogen gradient: an mRNA gradient dictates the protein gradient

The Bicoid (Bcd) protein gradient is generally believed to be established in pre-blastoderm Drosophila embryos by the diffusion of Bcd protein after translation of maternal mRNA, which serves as a strictly localized source of Bcd at the anterior pole. However, we previously published evidence that the Bcd gradient is preceded by a bcd mRNA gradient. Here, we have revisited and extended this observation by showing that the bcd mRNA and Bcd protein gradient profiles are virtually identical at all times. This confirms our previous conclusion that the Bcd gradient is produced by a bcd mRNA gradient rather than by diffusion. Based on our observation that bcd mRNA colocalizes with Staufen (Stau), we propose that the bcd mRNA gradient forms by a novel mechanism involving quasi-random active transport of a Stau-bcd mRNA complex through a nonpolar microtubular network, which confines the bcd mRNA to the cortex of the embryo.

Perlecan and Dystroglycan act at the basal side of the Drosophila follicular epithelium to maintain epithelial organization

Dystroglycan (Dg) is a widely expressed extracellular matrix (ECM) receptor required for muscle viability, synaptogenesis, basementmembrane formation and epithelial development. As an integral component of the Dystrophin-associated glycoprotein complex, Dg plays a central role in linking the ECM and the cytoskeleton. Disruption of this linkage in skeletal muscle leads to various types of muscular dystrophies. In epithelial cells, reduced expression of Dg is associated with increased invasiveness of cancer cells. We have previously shown that Dg is required for epithelial cell polarity in Drosophila, but the mechanisms of this polarizing activity and upstream/downstream components are largely unknown. Using the Drosophila follicle-cell epithelium (FCE) as a model system, we show that the ECM molecule Perlecan (Pcan) is required for maintenance of epithelial-cell polarity. Follicle cells that lack Pcan develop polarity defects similar to those of Dg mutant cells. Furthermore, Dg depends on Pcan but not on Laminin A for its localization in the basal-cell membrane, and the two proteins bind in vitro. Interestingly, the Dg form that interacts with Pcan in the FCE lacks the mucin-like domain, which is thought to be essential for Dg ligand binding activity. Finally, we describe two examples of how Dg promotes the differentiation of the basal membrane domain: (1) by recruiting/anchoring the cytoplasmic protein Dystrophin; and (2) by excluding the transmembrane protein Neurexin. We suggest that the interaction of Pcan and Dg at the basal side of the epithelium promotes basal membrane differentiation and is required for maintenance of cell polarity in the FCE.

Epithelial synthesis of tenascin at tips of growing bronchi and graded accumulation in basement membrane and mesenchyme

The extracellular matrix protein, tenascin, has been proposed as mediator in epithelial-mesenchymal interactions because of its characteristic distribution during embryogenesis. Here we compared the accumulation of tenascin and laminin in the early chicken lung bud. Laminin is deposited in the basement membrane, starting at the tips and increasing along the shafts of growing primary and secondary bronchi. In contrast, tenascin accumulation is highest in basement membranes and mesenchyme at sites where new bronchial branches are formed. By in situ hybridization, tenascin mRNA was found to be produced exclusively by the epithelium at sites of active growth of bronchial tubes.

Dally-like protein, a new Drosophila glypican with expression overlapping with wingless.

Proteoglycans, the molecules of extracellular matrix, carry a highly negative charge due to their glycosaminoglycan (GAG) chains and large volumes. They were considered to play a secondary role in activities like cell division, adhesion, blood coagulation, etc. until the importance of their sugar chains in the fibroblast growth factor (FGF) signalling was discovered (Science 252 (1991) 1705; Cell 64 (1991) 841). Studies of mutations in the genes sugarless(sgl) and sulfateless (sfl) have proved that the proteoglycans involved in Wg signalling contain heparan sulfate GAG chains (Development 124 (1997) 2623; Development 124 (1997) 3055; Development 124 (1997) 3565; Development 126 (1999) 3715). This has led to the attribution of specific functions to these molecules (J. Cell Biol. 148 (2000) 227). The Glypican family of heparan sulfate proteoglycans (HSPGs) is characterized by core proteins with conserved cysteine residues and attachment to the cell surface by a glycosylphosphatidyl inositol (GPI) anchor. This may lead to endocytic pathways that are different from other HSPGs, higher lateral mobility and possible apical localisation in a cell (Proc. Natl. Acad. Sci, USA 85 (1988) 9557). Variations in their HS contents may effect binding properties and localisation (J. Cell Biol. 124 (1994) 149; J. Cell Biol. 132 (1996) 487), thus specialising each member for a unique biological function. Glypicans play important roles in morphogenetic pathways, e.g. human glypican 3 (GPC3) is mutated in Simpson-Golabi-Behmel syndrome making an individual prone to tumours (Nat. Genet. 12 (1996) 241). Dally, the first Drosophila member of the family, is essential for the wingless and decapentaplegic signalling pathways (Development 121 (1995) 3687; Development 124 (1997) 4113). Here, we report a new Drosophila glypican, dally-like protein (dlp) with all the features of a glypican. Based on expression studies we report its colocalisation with Wg.