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BIOL 303 Study Questions Set 5 and 6

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University of Waterloo
BIOL 303
Dragana Miskovic

BIOL 303 Study Questions Set 5 and 6 1. In Drosophila the initial events of development occur without fertilization – What is this process called and what happens during this process?  Drosophila eggs are activated as they pass through oviduct, independent of fertilization.  An influx of calcium ions causes the completion of meiosis, initiation of mRNA translation, cross-linking of vitelline envelope, and egg changes from flaccid to turgid. 2. Does Drosophila have a cortical granule reaction as we have seen in other systems? How might polyspermy be prevented in Drosophila?  There are no cortical granules  Micropyle is single site of sperm entry. The prevention of polyspermy is the physical blockage of only one sperm being able to go in at a time.  Females retain the sperm; so one mating is good for a lifetime of fertilization. 3. What is the difference between the syncytial, syncytial blastoderm, and cellular blastoderm stages of Drosophila development?  Syncytium: a single cell or cytoplasmic mass containing several nuclei, formed by fusion of cells or by division of nuclei. Karyokinesis without cytokinesis forms the multi-nucleated cell.  Syncytial blastoderm: In insects, the stage of blastoderm preceding the formation of cell membranes around the individual nuclei of the early embryo. Nuclei migrate to cortex to undergo three more cortical divisions.  Cellular blastoderm: the stage at which the blastoderm has gone from being a syncytium to a being cellular. Cell membranes grow inward between the peripheral nuclei, separating off individual cells from the inner yolky cytoplasm. 4. What are the pole cells and where/when do they form?  The first mononucleate cells after the rapid nuclear cell division to create the syncytium are created at the posterior pole, where the polar granules are tethered. These cells are called pole cells, and they will form the fly's germ line. They are cellularized before all other cells. 5. Does the first transcription of zygotic genes occur before or after cellularization?  Cycle 11: first zygotic gene transcription. Some is needed for cellularization.  Cycle 14: cellularization starts (microtubules first, then actin microfilaments) 6. What events are associated with the mid-blastula transition in Drosophila?  Mid-blasatula transition: is a stage during the blastula stage of embryonic development in which zygotic gene transcription is activated.  Associated with the slowing of nuclear division, cellularization, and increase in new RNA transcription 7. What influences the timing and onset of the mid-blastula transition in Drosophila?  The ratio of chromatin to cytoplasm: increasing amount of DNA in a constant amount of cytoplasm may dilute repressors.  The Smaug protein (RNA binding protein) and targeted degradation of maternally loaded mRNAs  The Zelda protein (maternally encoded transcription factor) and activation of zygotic genes 8. What morphological features are associated with the initial stages of gastrulation in Drosophila? What germ line related fates (ectoderm, mesoderm, endoderm) are associated with these features?  Mesoderm forms from the ventral furrow, pinches off and becomes a tube  Endoderm invagination: anterior and posterior mid-gut invaginations. Pole cells migrate into posterior mid-gut invagination.  Cephalic furrow forms. Drosophila Gastrulation (con’t)  ectoderm and mesoderm cells undergo convergence and extension – movement toward posterior and ventral midline, collectively known as germ band.  The germ band extends, the tail is pushed to lie behind the head at the dorsal side.  During germ band extension, organogenesis begins. Segmentation becomes apparent and precursors are specified.  The nervous system forms from the ectoderm.  Tail is brought back to the posterior position of embryo about halfway through embryogenesis. This is called germ band retraction.  This leaves a hole in the dorsal epidermis that is covered by an extra-embryonic tissue called amnioserosa (later dies during development, similar idea to placenta).  Dorsal lateral epidermal sheets spread to meet along midline, known as dorsal closure Read the following if you are puzzled by cephalic furrow: “During Drosophila gastrulation, the embryo is transformed from a regular array of morphologically identical cells, organized in a single layer, into a complex array of cell groups. These groups change cell shape and move together in a characteristic, predetermined manner. Two invaginations, the ventral and cephalic furrows, represent the first morphological manifestations of cell fate and differentiation programs. The ventral furrow (VF) invaginates along most of the ventral midline, bringing the mesoderm and possibly part of the anterior endoderm primordium into the interior of the embryo. Cephalic furrow (CF) invagination takes place laterally on both sides of the embryo, near its anterior end. Unlike VF formation, the CF is only transient. At the completion of germ-band extension, all cells of the CF slowly unfold back onto the surface of the embryo to contribute to the ectoderm. Although the CF is a prominent morphological feature of the early gastrula, its developmental role remains enigmatic. No mutations have been isolated that affect only the CF and the mutations known to affect the VF and posterior midgut (PMG) invaginations leave the CF unaffected. The cellular and genetic mechanisms that control its formation are also unknown. The absence of a CF in embryos derived from mothers mutant for bicoid and the reproducible shifts in its position and/or lateral extent observed in anteroposterior and dorsoventral pattern indicates that the cell shape changes respond directly to positional information. However, these observations did not provide a specific clue to how that positional information is translated into specific changes in cellular morphology.” 9. You should be able to: Draw and label a mid-stage Drosophila egg chamber, indicating the nurse cells, the oocyte, the follicle cells, and the underlying orientation of microtubules within the egg chamber.  Nurse cells: provide nutrients the embryo will require, storing them in the yolk. Nurse cells surround the developing oocyte and synthesize proteins and RNAs that are to be deposited in it. 10. The developing egg chamber establishes an anterior/posterior axis mid-way through oogenesis. How does this happen and what observations demonstrate this polarity? (ie observations made following in situ hybridization)  The localization of bicoid, oskar and gurken mRNAs in the Drosophila oocyte defines the anterior-posterior and dorsal-ventral axes of the embryo.  Anterior: Bicoid mRNA localizes to anterior of the oocyte, directing where head and thorax of embryo develop.  Posterior: Posterior localisation of oskar mRNA directs formation of the pole plasm that contains the abdominal and germline determinants.  Dorsal: Gurken is translated on the dorsal side, producing a signalling molecule that causes adjacent follicle cells to define the embryo's dorsal-ventral axis.  Gurken mRNA is synthesized by nurse cells and transported to oocyte nucleus – gurken mRNA is localized between the oocyte nucleus and the oocyte membrane where it is translated. Follicle cells have a receptor for Gurken (epidermal growth factor, receptor called torpedo). The cells receiving the signal from Gurken are posteriorized, then send a signal back to oocyte. The signal recruits Par-1 protein to posterior edge of cytoplasm and organizes microtubules such that their plus ends are at the posterior end.  Par-1 is a serine/threonine kinase that has a role in cell polarity.  Now that microtubules are set up with plus ends at the posterior side, molecules can be localized through association with motor proteins. 11. The developing oocyte posterior initially localizes oskar mRNA, but later we observe an accumulation of nanos mRNA. What is the relationship or mechanism by which this happens?  Oskar mRNA is transported by a kinesin to posterior oocyte. Only in this area can oskar be translated. Oskar protein recruits more Par-1 to reinforce microtubule arrangement.  Posterior pole is now distinctive. It contains determinants for producing abdominal segments and germ cell determinants.  Different motor proteins (dyneins & kinesins) will move either towards the microtubule minus end (all dyneins) or plus end (most kinesins). Cargo tethered to specific motor proteins can, therefore, be delivered to either the anterior or posterior of the developing oocyte  Using this general mechanism of microtubule associated motor transport, two key determinants are also set up at this time: Bicoid mRNA is localized to the oocyte anterior. Nanos mRNA (because it is bound by Oskar protein) is localized to the oocyte posterior. 12. What type of product is encoded by the Gurken gene? It is frequently said that “Gurken is used twice” during oogenesis. Explain. • As oocyte grows the oocyte nucleus moves to an anterior dorsal position. • Gurken is again used – localized expression between the oocyte nucleus and oocyte membrane forms a gradient • Follicle cells nearby are dorsalized by the gurken signal • Gurken signals from the oocyte to the adjacent follicle cells twice during Drosophila oogenesis; first to induce posterior fate, thereby polarizing the anterior-posterior axis of the future embryo and then to induce dorsal fate and polarize the dorsal-ventral axis. Here we show that Gurken induces two different follicle cell fates because the follicle cells at the termini of the egg chamber differ in their competence to respond to Gurken from the main-body follicle cells in between. 13. Maternal effect mutations of the torpedo and Gurken genes produce very similar phenotypes where the follicles calls and the resulting embryos are ventralized/dorsalized.  2 different mutants  Maternal mutants of gurken cause ventralization of eggs & embryos (there is no dorsal specification); it is also true of torpedo.  Gurken mutation and Torpedo mutation giving similar phenotypes  Gurken is active only in oocyte (germ line cells) and torpedo is active only in follicle cells (somatic cells)  Why are these genes required: Germ line is polarized. Germ line goes on to make nurse cells and oocyte. Follicle cells derived from soma.  Gurken signals from the oocyte to the adjacent follicle cells twice during Drosophila oogenesis; first to induce posterior fate, thereby polarising the anterior-posterior axis of the future embryo and then to induce dorsal fate and polarise the dorsal-ventral axis. Here we show that Gurken induces two different follicle cell fates because the follicle cells at the termini of the egg chamber differ in their competence to respond to Gurken from the main-body follicle cells in between  Gurken is required twice during oogenesis to induce fate changes in the follicular epithelium. During early stages of oogenesis Gurken signaling from the posteriorly localized oocyte instructs the follicle cells in contact with the oocyte to adopt a posterior fate. Later, as the germinal vesicle assumes an asymmetric position at the anterior cortex of the oocyte, gurken mRNA and protein become tightly localized to its vicinity. Gurken signaling will then induce dorsal fates in the adjacent follicle cells. 14. Before Gurken and torpedo were cloned and identified as homologs of _______ and _______, respectively, a key experiment was performed that predicted how Gurken and Torpedo might be functioning. Briefly describe this key experiment, the observed result and the interpretation.  Gurken = ECF  Torpedo – ECFR  Experiment: pole cell transplantation. It established that torpedo was not needed in the germ line but was required in the somatic follicle cells for proper dorsalization of egg & embryo. 15. The activity of Gurken and Torpedo during oogenesis results in a dramatic gradient of nuclear localization of the transcription factor Dorsal at the syncytial blastoderm stage of development. Describe the general pathway
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