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Axis Specification and Patterning I
Syncytial specification in the
Drosophila embryo
We know more about the genetics of Drosophila melanogaster
than any other multicellular organism thanks to the pioneering
studies initiated by Thomas Hunt Morgan
But Drosophila proved to be a difficult organism to study embryology.
The Drosophila embryo was neither transparent so that it could be observed
microscopically nor was it large enough to be manipulated experimentally.
It was not until the advent of molecular biology, which facilitated the
identification and modification of genes and RNAs did it become possible to
relate the genetics of Drosophila to its development
Fertilization
Cleavage
http://www.wisegeek.com/what-is-the-fertilization-process.htm
Specification of early embryos happen by cytoplasmic determinants stored in the oocyte.
Cell membranes formed during cleavage partition the cytoplasm into blastomeres.
In Drosophila cell membranes do not form till after the 13th nuclear division
Figure 6.2 Laser confocal micrographs of stained chromatin showing syncytial
nuclear divisions and superficial cleavage in a series of Drosophila embryos
The specification of the cell types along the anterior-posterior and dorsal-ventral
axes is accomplished by interactions of components within the multinucleated cell
http://willy.supatto.perso.sfr.fr/3dentier.gif
Axial differences are initiated at an earlier stage by position of the egg within the egg chamber
Unlike worms where sperm entry site fixes the axes, in drosophila the interactions between the
oocyte and the follicle cells in the egg chamber before fertilization fix the axes
Genes the pattern the Drosophila body plan
A powerful “forward genetics” approach in the early 1990s spearheaded by Christiane Nϋsslein-Volhard and Eric Weischaus
was employed to identify the genes involved in shaping the larval and adult Drosophila. Randomly mutagenized flies were
screened for mutations that disrupted the normal body plan.
http://biology.kenyon.edu/courses/biol114/Chap13/Chapter_13A.html
The genes involved in these mutant phenotypes were cloned and characterized by many different groups worldwide
Primary axis formation………….it all begins during oogenesis
A single female germ cell the
oogonium divides four times with
incomplete cytokinesis to give 16
cells that are interconnected. One
of these will become the oocyte
and the rest will be nurse cells.
These are surrounded by somatic
cells called follicles cells.
The oocyte, nurse cells and the
surrounding somatic follicle cells
form the egg chamber.
http://www.zoology.ubc.ca/~bio463/lecture_13.htm
Anterior-posterior polarity in the oocyte
The follicle cells surrounding the oocyte are initially uniform but this is broken by two
signals organized by the oocyte nucleus involving the same gene gurken
The gurken RNA is made in nurse cells but transported to the oocyte and localized specifically between the
posteriorly localized oocyte nucleus and the follicle cells. The Gurken protein is translated and activates the Torpedo
receptors on the follicle cells giving them a posterior identity.
The posterior follicle cells send a
unknown signal back to the oocyte
which brings the Par-1 protein to
the posterior edge of cytoplasm
(Green signal in C).
Par-1 organizes the microtubules
such that the minus end is located
to the anterior and the plus end to
the posterior.
This microtubule orientation is
critical as different end-directed
motors such as kinesins and
dyneins will target their mRNA and
protein cargoes to different ends
thus establishing the anterior and
posterior axis of the oocyte.
The cytoskeletal
rearrangements lead to
localization of maternal
messages such as
bicoid and nanos mRNAs.
Also the oocyte volume
increases and the oocyte
nucleus is pushed by
growing microtubules to an
anterior-dorsal location.
Dorsal-ventral polarity in the oocyte
The gurken message along with the nucleus gets localized in a crescent
shape to the dorsal-anterior corner. The Gurken protein then signals
through torpedo to the overlying follicle cells to adopt a dorsal-follicle fate.
Maternal deficiencies in either the gurken or torpedo gene causes ventralization of the
egg chamber and also the embryo that develops from this egg.
Gurken is required in the oocyte and torpedo in the follicle cells shown elegantly by
making germ-line/somatic chimeras by Trudi Shϋpbach and colleagues in 1987
Reciprocal interactions between the oocyte
and egg chamber (follicle cells) specify the
dorsal-ventral polarity of the embryo
Establishment of dorsal-ventral polarity of the egg
chamber (follicle cells)
Establishment of
dorsal-ventral polarity
of the embryo
Cell. 1992 Feb 7;68(3):429-40.
Multiple extracellular activities in Drosophila egg perivitelline fluid are
required for establishment of embryonic dorsal-ventral polarity.
Stein D1, Nüsslein-Volhard C.
Embryonic dorsal-ventral polarity is defined within the perivitelline compartment surrounding the embryo by
the ventral formation of a ligand for the Toll receptor.
Here (as demonstrated by the transplantation of perivitelline fluid) are found three separate activities present in
the perivitelline fluid that can restore dorsal-ventral polarity to mutant easter, snake, and spatzle embryos,
respectively.
These activities are not capable of defining the polarity of the dorsal-ventral axis; instead they restore structures
according to the intrinsic dorsal-ventral polarity of the mutant embryos. They appear to be involved in the
ventral formation of a ligand for the Toll protein.
This process requires serine proteolytic activity; the injection of serine protease inhibitors into the perivitelline
space of wild-type embryos results in the formation of dorsalized embryos (Stein, 1992).
Injection experiments involving the use of dominant active Easter (Chasan, 1992) and Snake, as well as injection
of perivitelline (PV) fluid from dorsal mutant embryos into gd mutant embryos, have lead to production of ventral
elements at the site of injection, rather than in the normal ventral region (Stein, 1992).
These data suggest that D/V polarity is established by asymmetric presentation of the Toll ligand to the oocyte.
PV fluid from dorsal mutant embryos (thought to be depleted of Spätzle ligand because of the presence of the
Toll receptor) can rescue D/V polarity in snake and easter mutant embryos. This same PV fluid cannot restore
normal ventral structures to gd embryos.
In contrast, injection of PV fluid from Toll mutant embryos (thought to contain active Spätzle ligand) into gd
embryos produces ventral structures at the site of injection (Stein, 1992). The same fluid injected into snake or
easter embryos produces embryos with normal polarity, independent of the site of injection (Stein, 1992).
Generation of Dorsal-ventral pattern in the embryo by Dorsal the morphogen
16 cells with the highest concentration of dorsal
are the ones that generate the mesoderm.