10. Oogenesis
We then dissect ovaries from the created fly and use the egg chambers from stages in midoogenesis to visualize endogenous oskar mRNA via fluorescence in situ hybridization (FISH) - technique used to detect and localize the presence or absence of specific nucleic acids(mRNAin our case) sequences . FISH uses fluorescent probes that bind to only those parts of the mRNA with which they are complementary to. Here we use fluorescent probes named molecular beacons. 10 10

11. Localization of maternal mRNAs
gurken mRNA
bicoid mRNA
oskar mRNA
Since mRNA transport and localization processes are most prominent before fertilization, these mechanisms are best studied during oogenesis.
In Drosophila, the two ovaries are composed of strings of maturing egg chambers whose development spans over more than 80 hours and is divided into 14 morphologically distinct stages.
During the early stages, 4 cellular divisions occur, giving rise to an egg chamber consisting of 16 cells enveloped in a monolayer of somatically derived cells, called follicle cells.
By stage 6, the posterior most cell, becomes distinguished as the oocyte and the rest becoming the nurse cells that are connected to each other and the oocyte via a system of channels called ring canals.
These cytoplasmic bridges allow the intercellular flow of molecules and organelles into the oocyte.
As the oocyte grows, the follicle cells migrate to the posterior continuing to envelop the oocyte, until they disintegrate at later stages. Nurse cells disintegrate as well, leaving the developing oocyte to encompass the entire egg chamber.
The mature egg is subsequently fertilized in utero and deposited.
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The majority of mRNAs are synthesized in the nurse cells throughout oogenesis and are then localized to specific regions within the oocyte.
As I mentioned before, gurken mRNA is among the first to localize.
After oocyte determination, gurken localizes around the nucleus at the posterior pole. Upon a signaling event, the nucleus migrates to the anterior-dorsal corner, and gurken mRNA concentrates between the nuclear envelope and the adjacent cortex where it is maintained throughout mid-oogenesis.
Another maternal mRNA that is localized early in oogenesis, is bicoid mRNA. After it’s synthesis in the nurse cells, bicoid is localized at the anterior cortex of the oocyte where it continues to accumulate throughout oogenesis. Its localization is required for the proper development of the head and thorax of the fly.
The third maternal gene responsible for establishing the body axis is oskar. Its mRNA is also synthesized in the nurse cells, and transported into the oocyte where it traverses the cytoplasm and anchors at the posterior pole in a tight thin sliver.
Oskar localization and translation are the first steps in a cascade of molecular localizations that result in the assembly of polar granules and finally result in the establishment of the germ line at the pole plasm.
The transport and localization processes of these mRNAs have been inferred from a combination of genetic analysis, biochemical approaches and several RNA detection techniques.
Molecular level??

15. Required for localization of oskar mRNA at the posterior
since it is known that staufen is necessary for oskar mRNA localization we use it as a negative control
Staufen protein co-localizes with oskar mRNA at the poterior end and is required for oskar mRNA transport process. It contains 5 domains one of which is an RNAbinding domain. There is no proof showing direct association between oskar mRNA and Staufen. The function of their colocalization still remains unknown.
Required for localization of oskar mRNA at the posterior
No proof of direct association between the two
Function of their co-localization remains unknown.
(anchoring protein? Translation de-repression?) 15