Establishment of dorsal-ventral polarity of the Drosophila egg requires capicua action in ovarian follicle cells.

The dorsal-ventral pattern of the Drosophila egg is established during oogenesis. Epidermal growth factor receptor (Egfr) signaling within the follicular epithelium is spatially regulated by the dorsally restricted distribution of its presumptive ligand, Gurken. As a consequence, pipe is transcribed in a broad ventral domain to initiate the Toll signaling pathway in the embryo, resulting in a gradient of Dorsal nuclear translocation. We show that expression of pipe RNA requires the action of fettucine (fet) in ovarian follicle cells. Loss of maternal fet activity produces a dorsalized eggshell and embryo. Although similar mutant phenotypes are observed with regulators of Egfr signaling, genetic analysis suggests that fet acts downstream of this event. The fet mutant phenotype is rescued by a transgene of capicua (cic), which encodes an HMG-box transcription factor. We show that Cic protein is initially expressed uniformly in ovarian follicle cell nuclei, and is subsequently downregulated on the dorsal side. Earlier studies described a requirement for cic in repressing zygotic target genes of both the torso and Toll pathways in the embryo. Our experiments reveal that cic controls dorsal-ventral patterning by regulating pipe expression in ovarian follicle cells, before its previously described role in interpreting the Dorsal gradient.

Spätzle regulates the shape of the Dorsal gradient in the Drosophila embryo.

Dorsal-ventral polarity of the Drosophila embryo is established by a nuclear gradient of Dorsal protein, generated by successive gurken-Egfr and spätzle-Toll signaling. Overexpression of extracellular Spätzle dramatically reshapes the Dorsal gradient: the normal single peak is broadened and then refined to two distinct peaks of nuclear Dorsal, to produce two ventral furrows. This partial axis duplication, which mimics the ventralized phenotype caused by reduced gurken-Egfr signaling, arises from events in the perivitelline fluid of the embryo and occurs at the level of Spätzle processing or Toll activation. The production of two Dorsal peaks is addressed by a model that invokes action of a diffusible inhibitor, which is proposed to normally regulate the slope of the Dorsal gradient.

Signaling pathways that establish the dorsal-ventral pattern of the Drosophila embryo.

The dorsal-ventral pattern of the Drosophila embryo is established by three sequential signaling pathways. Each pathway transmits spatial information by localizing the activity of an extracellular signal, which acts as a ligand for a broadly distributed transmembrane receptor. The components of the first two pathways are encoded by maternal effect genes, while the third pathway is specified by genes expressed in the zygote. During oogenesis, the oocyte transmits a signal to the surrounding follicle cells by the gurken-torpedo pathway. After fertilization, the initial asymmetry of the egg chamber is used by the spätzle-Toll pathway to generate within the embryo a nuclear gradient of the transcription factor Dorsal, which regulates the regional expression of a set of zygotic genes. On the dorsal side of the embryo, the decapentaplegic-punt/thick veins pathway then establishes patterning of the amnioserosa and dorsal ectoderm. Each pathway uses a distinct strategy to achieve spatial localization of signaling activity.

A processed form of the Spätzle protein defines dorsal-ventral polarity in the Drosophila embryo.

Stein et al. (1991) identified a soluble, extracellular factor that induces ventral structures at the site where it is injected in the extracellular space of the early Drosophila embryo. This factor, called polarizing activity, has the properties predicted for a ligand for the transmembrane receptor encoded by the Toll gene. Using a bioassay to follow activity, we purified a 24 x 10(3) M(r) protein that has polarizing activity. The purified protein is recognized by antibodies to the C-terminal half of the Spätzle protein, indicating that this polarizing activity is a product of the spätzle gene. The purified protein is smaller than the primary translation product of spätzle, suggesting that proteolytic processing of Spätzle on the ventral side of the embryo is required to generate the localized, active form of the protein.

The spätzle gene encodes a component of the extracellular signaling pathway establishing the dorsal-ventral pattern of the Drosophila embryo.

spätzle is a maternal effect gene required in the signal transduction pathway that establishes the dorsal-ventral pattern of the Drosophila embryo. spätzle acts immediately upstream of the membrane protein Toll in the genetic pathway, suggesting that spätzle could encode the ventrally localized ligand that activates the receptor activity of Toll. The spätzle gene encodes a novel secreted protein that appears to require activation by a proteolytic processing reaction, which is controlled by the genes that act upstream of spätzle in the genetic pathway. We propose that proteolytic processing of the spätzle protein is confined to the ventral side of the embryo and that the localization of processed spätzle determines where the receptor, Toll, is active.

Tn10 transposition and circle formation in vitro.

We describe a cell-free system that promotes Tn10 transposition and transposon circle formation, a related intramolecular event. Tn10 circle formation in vitro has been characterized in detail, and is shown to require a supercoiled substrate and to proceed in the absence of ATP. The reaction requires Tn10 transposase protein, and either of two E. coli proteins, integration host factor (IHF) and HU, which are small DNA binding proteins that change the conformation of DNA. Tn10 is composed of inverted repeats of insertion sequence IS10. Pair-wise combinations of the IS10 "outside" and "inside" ends mediate distinct classes of rearrangements in vivo, and they exhibit different reaction requirements in vitro. In contrast to the Tn10 reaction, which involves two outside ends, circle formation with two inside ends proceeds with a transposase fraction alone, in the absence of added host factors, and is inhibited by methylation of the dam site within each terminus.

New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition.

We describe below several new variants of the tetracycline-resistance transposon Tn10 which are more useful than the wild-type transposon for many types of genetic and physical analysis of bacteria. These derivatives have one or more of the following new properties: (i) new drug resistance markers; (ii) high transposition frequencies; (iii) removal of the transposase gene to a position outside of the transposing segment; (iv) internal deletions which eliminate the ability of Tn10 to make adjacent deletion/inversions; or (v) addition of a trp-lac operon fusion segment just inside one terminus such that insertion can automatically generate a transcriptional fusion to the interrupted operon. Phage and plasmid vehicles carrying these new elements are described.

Transposase promotes double strand breaks and single strand joints at Tn10 termini in vivo.

We present evidence that Tn10 transposase promotes double strand breaks and single strand joints at Tn10 termini in vivo. Plasmids containing a shortened Tn10 element and a transposase overproducer fusion give rise, upon transposase induction, to new DNA species. The most prominent class is a circularized transposon molecule whose structure suggests that it arises from double strand breakage at the two transposon ends followed by covalent joining between the 3' and 5' ends of one of the two strands. We have used formation of the circularized transposon as a physical assay for the interaction between transposase and different mutant and wild-type termini. These experiments show that transposase protein interacts preferentially with the genetically most active termini in a way that suppresses productive interaction with weaker termini present on the same substrate molecule.

Tn10 transposase acts preferentially on nearby transposon ends in vivo.

Transposition of Tn10 requires sites at the termini of the element and one essential transposon-encoded function, "transposase", which acts at those termini. Genetic complementation experiments reveal that this "transposase" function works much more efficiently on transposon ends located near the gene from which they are expressed than on transposon ends located at a distance. This property accounts for the failure of mutant Tn10 elements to be efficiently complemented in trans. The failure of transposase protein to move freely in three dimensions could be explained by one-dimensional diffusion, energy-dependent translocation and/or extreme protein lability. Additional genetic analyses demonstrate that the rate of Tn10 transposition in vivo depends upon the length of the transposon and the amount of transposase protein. Function dependence and length dependence are independent aspects of the transposition process that could correspond to the break/join and replication aspects into which transposition has been separated conceptually.

Effect of multimeric structure of the factor VIII/von Willebrand factor protein on binding to platelets.

The characteristics of the intact factor VIII/von Willebrand factor protein binding to human platelets was compared to 2-mercaptoethanol-treated factor VIII/von Willebrand factor protein and to fractions of plasma factor VIII/von Willebrand factor protein that elute after the void volume. These studies indicate that the factor VIII/von Willebrand factor protein larger size oligomers bind preferentially with high affinity to low capacity sites on human platelets. The intermediate and smaller size oligomers bind with intermediate or low affinity to sites with a much greater capacity. The results from binding analysis are also paralleled by the competitive inhibition of the intact factor VIII/von Willebrand factor protein by the various 2-mercaptoethanol-treated materials. These studies indicate that the two classes of binding sites seen in previous reports of factor VII/von Willebrand factor binding reflect heterogeneity in the oligomer size of the factor VIII/von Willebrand factor protein used in these assays. This study provides a model for understanding some of the normal structure-function relationships of the normal factor VIII/von Willebrand factor protein and the defect(s) in a variant form of von Willebrand's disease. In this form of the disease, decreased factor VIII/von Willebrand factor binding to platelets is reflected in decreased von Willebrand factor activity but coagulant and/or antigen levels are normal or only slightly decreased.

Selective binding of the factor VIII/von Willebrand factor protein to human platelets.

The factor VIII/von Willebrand factor protein was radiolabeled after modification by galactose oxidase and reduction with tritiated potassium borohydride. This mild efficient method for labeling resulted in retention of over 90% of the biologic activities of the factor VIII/von Willebrand factor protein. Binding of this protein to platelets was found to be specific, and binding sites could be saturated in the presence of ristocetin. However, binding was highly dependent on ristocetin concentration, as the number of human factor VIII/von Willebrand factor molecules bound per platelet was a function of the ristocetin concentration. At a ristocetin concentration of 0.55 mg/ml, each platelet binds approximately 11,000 factor VIII/von Willebrand factor molecules per platelet. Scatchard analysis of the concentration-dependent binding sites yielded a hyperbolic plot that appeared to be related to the existence of two classes of binding sites. The higher affinity class had a Kd of 3.7 x 10(-10) M 3500 sites/platelet, while the lower affinity class had a Kd of 2.35 x 10(-9) M and a capacity of 7500 sites/platelet. As with ristocetin-induced platelet agglutination, the carbohydrate content plays a significant role in the binding of the factor VIII/von Willebrand factor protein to the platelet.