October 9, 1996 TIME: 120 minutes
TOTAL POINTS: 20
1. Fill in the blanks (0.5 points each).
The issue of how the Xenopus egg develops dorsal-ventral polarity
during the cleavage stages has recently become clarified. Antisense oligo-knockout
experiments have shown that elimination of ß-catenin is sufficient
to eliminate the organizer (credit also given for Nieuwkoop center or
dorsal mesoderm or dorsal-ventral axis). Likewise, elevated ß-catenin
on the ventral side of a Xenopus embryo is sufficient to duplicate
the embryonic axis. During normal development, ß-catenin is found
in greater abundance on the dorsal side than on the ventral
side. One way by which the abundance of ß-catenin can be regulated
is by degradation, which is regulated by the process of phosphorylation
by Xgsk-3. According to this hypothesis, the dorsal-ventral differences
in ß-catenin abundance result in the formation of the region called
the Nieuwkoop center on the dorsal side of the embryo.
ß-catenin is homologous with armadillo in Drosophila,
whereas the protein that directly regulates it is homologous with shaggy.
These proteins are in the Wnt (or wingless) signal transduction cascade.
ß-catenin presumably functions by interacting with Xtcf-3 and regulating
transcription of target genes in the nucleus (either directly or indirectly).
The organizer is formed as a result of signals that emanate from the
The original dorsal-ventral asymmetry of the Xenopus embryo is caused by cortical rotation, which is triggered by entry of the sperm.
2. Describe how the synthesis and accumulation of 5S RNA are thought to be controlled during Xenopus oogenesis (2.5 points).
Transcription is regulated by the interaction of the transcription
factor TFIIIA with the internal promoter. After it is synthesized, the 5S
RNA binds to free TFIIIA, thus titrating it, resulting in its eventual depletion
and cessation of transcription.
3. Fill in the blanks (0.5 points each):
Both doral-ventral and anterior-posterior patterning of the Drosophila oocyte are dependent upon a signal that is encoded by the gurken gene. Precise localization of this signal depends upon redistribution of the gurken mRNA, which is a consequence of the function of microtubules. Once the anterior-posterior axis has been established,
bicoid mRNA becomes localized to the anterior end of the oocyte, whereas oskar mRNA becomes localized to the posterior end. The latter messenger is localized as the result of the function of microtubules.
Cite your evidence for the last statement (1.5 points):
oskar mRNA localization is abolished by treatment with colchicine.
What portion of this mRNA interacts with this entity (0.5 points)?
4. Provide clear definitions of the following terms (1.5 points each):
A. the zona reaction
The zona reaction is analogous to the cortical reaction in sea urchins. Hydrolytic enzymes released from the cortical granules into the perivitelline space harden the zona pellucida, making it refractory to sperm penetration and inactivating sperm receptors. The latter may involve a chemical modification to ZP-3, whereby sugar groups are hydrolyzed by glycosidases released from the cortical granules. The zona hardening mechanism is unknown, but it does not involve formation of glycoprotein cross-linkages.
MPF stands for Maturation Promotion Factor. MPF was first demonstrated
by Yoshio Masui, who demonstrated that oocytes could be caused to undergo
maturation by injecting them with cytoplasm from progesterone-treated eggs.
Serial transfers of cytoplasm from maturing recipients will continue to
induce maturation in recipients even though only the original donor was
exposed to progesterone. MPF activity has also been found in cells of cleaving
Xenopus embryos, oscillating with the period of the cell cycle, appearing
in late G2, peaking in metaphase and disappearing by interphase. MPF is
now known to be present with the same periodicity in a variety of cells
and is thus referred to as M-phase Promotion Factor.
MPF was shown by Lohka and Maller to be comprised of two subunits:
p34 (homologous to cdc2) and p45 (cyclin). Both of these constituents are
kinase enzymes. MPF pre-exists in the oocyte in an inactive form. Progesterone
triggers maturation by causing a reduction in the levels of cyclic AMP,
which initiates translation of c-mos mRNA. c-mos activates the cyclin component
of MPF by phosphorylation. The cyclin, in turn, activates p34 by phosphorylating
it. p34 phosphorylates a number of proteins, including lamins and histone
H1. Phosphorylation of lamins causes dissociation of the nuclear envelope,
and phosphorylation of histone H1 facilitates chromosome condensation. c-mos
also functions as cytostatic factor (CSF), which stabilizes cyclin and retaining
MPF activity until fertilization. In somatic cells, cyclin is degraded during
each cell cycle, which allows for progression through the cell cycles.
c. phospholipase C
An enzyme in the inositol triphosphate (IP3) pathway that catalyzes
the conversion of PIP2 into IP3 and diacylglycerol (DAG). Its activity is
activated by G protein in response to binding of sperm to a sperm receptor.
The IP3 is released into the cytoplasm, where it releases Ca++ from the
endoplasmic reticulum. The liberation of Ca++ triggers the cortical reaction
that produces the slow block to polyspermy, whereas DAG remains in the membrane
and activates the Na+/H+ pump that raises internal pH and triggers egg activation,
including activation of protein synthesis. Phospholipase C is bound to the
inner surface of the plasma membrane. Students note: A drawing of the IP3
pathway was helpful to demonstrate that you understood the relationships
among the various components:
d. Pole cells of Drosophila (define and describe their origin and formation).
Pole cells are the presumptive germ cells in Drosophila. The Drosophila egg is centrolecithal and undergoes superficial cleavage to form a syncytial blastoderm. However, at the posterior pole, a group of energids enter the pole plasm and complete cleavage furroews segregate the nuclei into distinct cells that will later form the germ cells.