\"The graded nuclear location of the transcription factor Dorsal along the dorso
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Question
"The graded nuclear location of the transcription factor Dorsal along the dorsoventral axis of the early Drosophila embryo provides positional information for the determination of different cell fates. Nuclear uptake of Dorsal depends on a complex signalling pathway comprising two parts: an extracellular pro- teolytic cascade transmits the dorsoventral polarity of the egg chamber to the early embryo and gener- ates a gradient of active Spätzle protein, the ligand of the receptor Toll; an intracellular cascade down- stream of Toll relays this graded signal to embryonic nuclei. The slope of the Dorsal gradient is not deter- mined by diffusion of extracellular or intracellular components from a local source, but results from self-organised patterning, in which positive and neg- ative feedback is essential to create and maintain the ratio of key factors at different levels, thereby establishing and stabilising the graded spatial infor- mation for Dorsal nuclear uptake." Curr Biol 15:R887.
Article: http://www.cell.com/current-biology/fulltext/S0960-9822(05)01226-1
1) Pipe is a heparin-sulfate 2-sulfotransferase involved in extracellular matrix modification. In the embryo, it is involved in the specification of the ventral domain/area of the embryo. How is Pipe thought to be involved in starting the protease cascade that transfers Spätzle to the Toll receptor?
Explanation / Answer
Dorsoventral asymmetry in the early Drosophila embryo is mostly under maternal control, Axis formation starts at the ventral side of the embryo and gradually transferred to the embryo by the Toll pathway.
During Drosophila embryogenesis, the Toll pathway has three different regions along the dorsoventral axis, leading to separation of the mesoderm and the neuroectoderm from the non-neurogenic (dorsal) ectoderm .
Dorsal ectoderm is patterned by the zygotically regulated Decapentaplegic (Dpp) pathway, ventralising information generation involves 17 maternally provided factors, The spatial cues for the embryonic dorsoventral axis originate during oogenesis in the follicle cell layer, a somatic, monolayered epithelium, which surrounds the oocyte–nurse cell complex.
Factors obtained from the follicle cells are intracellular proteins Pipe (Pip), Slalom (Sll) and Windbeutel (Wbl) and the secreted protease Nudel (Ndl). All these proteins are required to activate a cascade of proteases which are secreted as inactive precursors by the oocyte or the early embryo.
The protease cascade is modulated by several intrinsic positive and negative feedback loops, alos by the Easter inhibitor Serpin27A. The activation of the cascade reaches a highest point of development, by the generation of the morphogen Spatzle (Spz), which binds to the transmembrane receptor Toll (Tl).
Pipe modifies the unknown ECM component, which is secreted by the follicular cells and maintained within the extracellular space, surrounding the late oocyte and early embryo. Subsequently, an extracellular signal is generated within the region defined by the modified ECM component. This signal induces the nuclear transport of the transcription factor Dorsal.
The ventral domain of the embryo is specified by Pipe in the follicular epithelium. Windbeutel allows the export of Pipe from ER to Golgi. Slalom transports the sulfate donor PAPS into the Golgi. There it acts as substrate for the sulfotransferase reaction carried out by Pipe. The autocatalytically processed Nudel central protease domain either processes the ECM component modified by Pipe or directly triggers the protease cascade by the activation of GD. GD activates Snake, which than activates Easter to process Spatzle, the ligand for the Toll receptor.
Pipe expression at the ventral side of the developing oocyte is sufficient to induce axis formation in the embryo, because ectopic expression of pipe in the follicular epithelium activates the Toll pathway.
But expression of pipe is not sufficent to explain the shape of the Dorsal gradient. Pipe is expressed evenly in a domain surrounding around 40% of the circumference of the egg chamber with sharp lateral borders . During egg maturation, this domain maintains its size compared to the egg circumference The entire bell-shaped Dorsal gradient is 40% of the egg circumference is present within the domain of uniform pipe expression.
But in mutants of grk or egfr, pipe repression is compromised and the pipe domain expands. The Dorsal gradient expands as well as splits and acquires two peaks, one at each side of the ventral midline . This shows that pipe not only localise a source, from which a gradient formed by diffusion but also defines a ventral region of the egg. In which a complex patterning system leads to gradient formation by dynamic interactions of its components.
The pipe locus encodes ten related proteins, which show similarity to heparansulfate 2-O-sulfotransferases of vertebrates Only one protein isoforms is expressed in the ventral follicle cells, is responsible for dorsoventral axis formation. Other isoforms are required for the late morphogenesis of the salivary glands.
Pipe acts as a sulfotransferase. The universal sulfate donor in sulfotransferase reactions is 3´-phosphoadenosine 5´-phosphosulfate (PAPS). Both the PAPS synthetase and Slalom, which imports PAPS from the cytoplasm into the Golgi apparatus , are required for dorsoventral pattern formation. Pipe transfers sulfate groups to the carbohydrate moieties of an unknown protein or glycolipid
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