Analysis of cis-acting RNA sequences required for intracellular localisation of gurken,the Drosophila TGF alpha homologu

Humans, flies and other multicellular animals, consist of many specialised cell types, located at their correct positions in the body. How such complex structures originate from the first simple cell, which is the fertilised oocyte or egg, has been a central question in biology. In the fruitfly, Drosophila, the first asymmetries arise when messenger RNA (mRNA) molecules that encode the information to make proteins, localise in specific places within the oocyte, and therefore target proteins to the same parts of the cell. How such mRNAs become restricted in their distribution is poorly understood. We will study how one key mRNA, gurken (grk) becomes localised to the dorsal (back) half of the egg and embryo, making it different from the ventral (front) half. Flies missing the grk gene lay eggs missing dorsal structures.
We will use exciting new methods we have developed to make grk mRNA molecules emit red light in order to follow their localisation after injection into living fly oocytes and embryos (0.5mm long). We will image the cells of the oocyte and embryos using proteins that emit green light and define the signals in the mRNA that target it to its dorsal destination. We will also investigate what other genes are responsible for the localisation and how they achieve their function.
Grk is related to the human transforming growth factor alpha (TGFa), which plays an important role in human oocyte maturation. TGFa is mutated in a number of different human cancers and developmental diseases. Before we can hope to cure such diseases, we must first understand how these molecules work, but studying the function of TGFa in humans is hampered by ethical and technical difficulties. Much more is known about these processes in Drosophila, in which many of the basic processes of development have turned out to be surprisingly similar to humans. We therefore anticipate that our work will be applicable to humans and may lead in the long term to a better understanding of how to cure diseases associated with malfunction of TGFa.

Intracellular mRNA localisation targets proteins to their cytoplasmic site of function and is important in many symmetry breaking processes ranging from yeast mating type switching and actin based cell motility, to embryonic axis specification in Drosophila and Xenopus. We have been studying the mechanism of asymmetric localisation of Drosophila gurken (grk), a TGFa homologue that plays a central role in the specification of the major body axes. grk mRNA is localised to one side of the oocyte nucleus, restricting the distribution of the Grk signal that leads to the establishment of the antero-posterior and the dorso-ventral axes of the oocyte and the embryo. The mechanism of grk mRNA localisation remains poorly understood largely because of the lack of rapid in vivo and in vitro assays for localisation. We have developed a novel and rapid in vivo assay by injecting fluorescently labeled grk RNA and imaging its assembly into particles that localise correctly in living oocytes and embryos (Appendix I). The assay will allow us to determine the mechanism and route of localisation. We will also define systematically the cis-acting sequences and trans-acting factors necessary and sufficient for grk mRNA particle formation and localisation. We have also been developing an in vitro assay for binding of grk RNA to microtubules and motility to their minus ends, using ovary or embryonic extracts added to fluorescent RNA and in vitro assembled microtubule asters (Appendix II). We will use the assay to demonstrate and study microtubule based motility and purify trans-acting factors that bind to specific cis-acting elements. In June 2000, I began a 5 year Lister Senior Fellowship.