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Development of Drosophila
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Hand-out CBT Chapter 2 Wolpert, 5 edition
October 2016
General
Drosophila melanogaster, the fruit fly, is found in all warm countries. In cooler regions, it is established by
migrants during the summer or can overwinter in warm places. It has been used for genetic experiments since
1909, due to its many advantages:
• simple and cheap breeding
• short life cycle
• high number of offspring
Within just one day the fertilized
egg develops from a single cell
into a complete and autonomous
organism consisting of many
different organs. It is the first 3
hours of this day that we will
study: the early egg stages.
There are 17 stages distinguished
in the development of the fruit fly
egg. This module will only look at
the first five stages, until the onset
of gastrulation.
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Stage
1
0:00-0:25
25 min
2
0:25-1:05
40 min
3
developmental activity
Pronuclear fusion and first two nuclear divisions.
A few hours after fertilization the egg is laid. Stage 1 begins once the egg has been
laid. It ends when the first two cleavage divisions have been completed. The cell
has now 4 nuclei. The image is from an egg after the first division, so there are only
two nuclei.
Syncytial divisions 3-8
During this stage cleavage
divisions 3 - 8 take place. A
characteristic of this stage is a
significant retraction of the embryo
from the vitelline envelope. This
leads to the appearance of two
empty spaces (perivitelline space),
one at the anterior and one at the
posterior pole of the egg.
During divisions 3 to 5, the nuclei
and their surrounding cytoplasm
tend to move posteriorly; by the
fifth cleavage, zygotic nuclei
occupy an ellipsoid field in the
center of the egg, between 20 and
80% egg length (0% egg length =
posterior pole). After this fifth
division there are 32 nuclei
present.
stage 2b
stage 2c
From the fifth
division on the
nuclei begin to
move
peripherally.
Some nuclei
remain centrally
located, others
drop out from the
periphery and
give rise to the
vitellophages
(yolk nuclei).
By the end of the eighth
division, the majority of
nuclei are evenly
arranged on an ellipse. At
the end of this stage
about 200 nuclei are
populating the periphery
and about 50
presumptive yolk nuclei
are located in the center
of the embryo. The
function of these yolk
nuclei is not fully
understood, and will not
be dealt with in this
module.
Syncytial division 9
1:05-1:20
15 min
Nuclear division 9 and polar bud formation take place during this stage. Polar bud
formation and the role of polar buds will be ignored in this module. At the end of this
stage there are about 512 nuclei.
4
Syncytial divisions: 10-13, end of cleavage divisions
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It is the syncytial blastoderm stage in which blastoderm nuclei perform the last four
cleavage divisions (10th, 11th, 12th and 13th division). The duration of cleavage
divisions 10-13 increases progressively, from approximately 8 min to 20 min. Stage
4 terminates at the beginning of cellularization. At that time there are over 8000
nuclei.
Cellularization of the blastoderm, cytoplasmic bridges still connect cells
1:20-2:10
50 min
2:10-2:50
40 min
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2:50-3:00
10 min
Cellularization occurs by means of the introgression of
membrane furrows to separate single blastoderm nuclei. This
is a rapid process, and is accomplished within 30 min at
25°C.
Blastoderm cells around the perimeter of the entire egg at
this stage are not completely isolated since they still maintain
connected with the syncytial yolk cytoplasm through wide
cytoplasmic bridges. These connections are lost during gastrulation.
Onset of gastrulation to form mesoderm and endoderm, cytoplasmic bridges are
lost
It is the stage of early gastrulation, in which the major morphogenetic movements
start:
• The invagination of the mesoderm is initiated.
• The blastoderm cells at the posterior pole form a dorsal plate underneath the
pole cells.
• The cephalic furrow becomes visible.
Gastrulation will not be dealt with in this module. It is covered in the module
'Gastrulation'.
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1. A. In which stage is the dorso-ventral axis determined?
B. In which stage is the anterior-posterior axis determined?
C. In which stage are the parasegments formed?
D. In which stage does segment formation start?
E. In which stages are maternal gene products involved?
2. There are four groups of genes or gene products that lead to the
subdivision of the fly embryo into segmental entities. Put the gene
groups in the order in which they effect the forming of
segmentation in Drosophila.
3. To which group do these genes belong?
bicoid
wingless
even-skipped
giant
Krüppel
4. The genes have effect in a certain order, and influence the expression of each other. This gives rise to a
certain hierarchy between the genes.
A. Determine the hierarchy of the genes A, B and C by mutant analysis. Hierarchy:
Embryo
View expression of A
View expression of B
View expression of C
Wildtype
Mutant A
Mutant B
Mutant C
B. Determine whether the interactions are predominantly activating or repressing.
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5. A. Which group(s) of genes encode mostly transcription factors?
o maternal genes
o gap genes
o pair-rule genes
o segment polarity genes
B. What is the reason segment polarity genes are not always transcription factor genes?
o transcription factors can not pass the plasma membrane
o transcription factors do not influence protein levels directly
o transcription factors need zygotic genes to activate
Maternal genes
6. A. What is the correct definition of maternal factors in Drosophila?
o The genes of the mother that affect the offspring, but not herself.
o The genes that are expressed by the follicle cells during oogenesis.
o The genes that are expressed by all cells involved during oogenesis.
B. Which cells can produce maternal factors?
o somatic cells of the mother
o follicle cells
o oocyte
o zygote
o nurse cells
o cells of the embryo
7. Maternal genes are the genes of the Drosophila mother fly that lays the egg. These genes have no effect in
the mother fly, but only in the egg it lays. Examples of maternal genes are bicoid and nanos.
A. Which is present in the Drosophila egg that influences its development?
o the gene itself
o the resulting mRNA
o the protein products of the gene
o the unspliced pre-mRNA
B. Where is the mRNA of bicoid and nanos localized in the egg?
C. How is it kept there?
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8. Drosophila has maternal genes which determine the posterior-anterior axis and genes which determine the
dorsal-ventral polarity. How many maternal genes for the posterior-anterior axis have (so far) been
discovered?
9. There are maternal genes which determine the anterior-posterior axis and genes which determine the
dorsal-ventral polarity in the larva. The genes for the anterior-posterior axis are divided in three groups:
Group
mutations effect
example
anterior
anterior region
bicoid
posterior
posterior region
nanos
terminal
both terminal regions
torso
These larvae are the result of a mutation in one of these genes. Which gene is most probably mutated in
each of these larvae?
10. In the bicoid mutant embryo bicoid is absent. There are several experiments you could perform with these
mutant eggs, in which you inject them at the anterior end with different materials. Which experiment has the
best chance of the embryo developing normally after fertilization?
o bicoid protein
o bicoid mRNA
o transcription factors of the bicoid gene
o none of these
11. In this unfertilized egg nanos mRNA is present at the posterior end. Nanos helps to establish an anteriorposterior gradient of the Hunchback protein.
A. What are the levels of the hunchback
mRNA in this Drosophila egg?
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B. Which of the following mechanisms by which Nanos influences the Hunchback protein gradient are
possible?
o prevents translation of the hunchback mRNA
o breaks down Hunchback protein
o breaks down hunchback mRNA
o prevents transcription of hunchback gene
C. What are the levels of the maternal nanos mRNA after fertilization?
.
12. Torso is also a maternal gene, but affects both anterior and posterior end.
A. What type of protein is the Torso protein?
B. The Torso protein is a tyrosine-kinase receptor, present in the plasma membrane. What are the levels
of this receptor in a fertilized Drosophila egg plasma membrane?
C. Why do these receptors only affect the terminal regions of the embryo?
o The ligand for the receptor is not distributed evenly
o The parts of the signalling pathway within the cell are not distributed evenly
o The receptors are only free to move through the membrane at the terminal ends, so only there
dimerization can take place
D. Receptors in the membrane and ligand in the vitelline envelope are both present before fertilization.
Why are these receptors not activated before fertilization?
o The ligand is immobilized in the vitelline envelope
o The receptors are immobilized by the vitelline envelope
o The ligand and receptors are separated by the vitelline envelope
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Dorso-ventral axis
13. The dorso-ventral axis is determined at the same
time as the anterior-posterior axis. There are a lot of
proteins involved in this process. Only one of these
protein gene products is not distributed evenly the
egg, and therefore responsible for the existence of
the axis.
The Dorsal protein is a transcription factor, which has
to go into the nucleus to activate and repress
transcription of certain genes involved in dorsoventral polarity.
Dorsal is initially bound to cactus, which prevents it
from entering the nucleus.
To break down Cactus a signalling pathway is
involved. The receptors are formed by the Toll
protein.
The ligand for this receptor is the Spätzle fragment.
This, however, is part of the Spätzle protein, and has to be separated before it can act as a ligand.
The Spätzle protein is processed by a chain of interactions, including the enzyme produced by the pipe
gene.
When the receptor is activated, it binds to the Tube protein, which in its turn activates Pelle, which activates
Tube.
Tube and Pelle together are able to break down the Cactus protein, thus allowing Dorsal to enter the
nucleus.
A. Which of these proteins is responsible for the dorso-ventral axis?
B. Which of these genes are maternal genes?
C. Where is pipe expressed in the embryotic body?
14. Dorsal enters the nuclei at the ventral side of the embryo. There it activates or suppresses
several genes.
A. What is the direct influence of Dorsal on the expression of these genes?
B. By what mechanism does Dorsal influence these genes?
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C. Dorsal is present in a gradient from the ventral side. Dorsal above a certain level activates the
expression of snail. Rhomboid is already activated at very low levels of Dorsal, but is suppressed by
Snail. What will the levels of Rhomboid be in this Drosophila embryo?
Oogenesis
15. In the ovarium of Drosophila the eggs are formed. An egg that is laid consists not only of
the oocyte, but hundreds of other cells.
At the right you see one of the two ovaries of a female fruit fly. In red is one of the 16
ovarioles, with at the top the germarium, and at the bottom a ripe oocyte.
Finish the following sentences:
The oocyte is formed from . . . . . . . . . . . . . . by . . . . . . . . . . . . . divisions.
The resulting . . . other cells form the . . . . . . . . . . . . . . . . . .
The oocyte and these nurse cells are surrounded by . . .
...................
16. In the ovarium of Drosophila the eggs are formed. An egg that is laid consists not only of the oocyte, but
hundreds of other cells.
A. Indicate the oocyte in the last four stages of the egg.
B. The oocyte moves towards the posterior end of the follicle. Which protein plays an important role in
this?
C. Indicate the cells in the last stage that are haploid (n).
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17. During oogenesis the oocyte moves to one end of the egg chamber. There its interaction with the follicle
cells initiates their specification as posterior polar follicle cells. Gurken in the oocyte is the responsible
protein.
A. Gurken is a:
o maternal gene
o zygotic gene
B. Where is Gurken protein transported to?
o it stays in the cytoplasm
o it ends up in the membrane of the oocyte
o it is secreted in the perivitelline space
C. What kind of protein is Gurken?
o receptor
o ligand
o second messenger
D. What is the name of this receptor?
18. During oogenesis the gradient for (among others) bicoid and oskar mRNA is formed, which will determine
the anterior-posterior axis. Where are bicoid and oskar mRNA's produced?
o oocyte
o nurse cells
o follicle cells
19. This oocyte is stained for bicoid mRNA. The bicoid mRNA is not dispersed
evenly throughout the cell.
A. What is causing this?
B. This oocyte is stained for bicoid mRNA, which is bound to the cytoskeleton. It will reorganize in such a
way that the bicoid mRNA will be at the anterior end, and oskar mRNA at the posterior end. This
process starts when the oocyte gets a signal. Where does this signal come from?
o posterior polar follicle cells
o dorsal follicle cells
o fertilization
o nucleus of oocyte
o nurse cells
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20. This is a picture of an ovariole of Drosophila.
A. When the egg is fertilized, what will be the
anterior side of the embryo?
B. The nucleus of the oocyt in the last stage has already moved to one side, which determines the ventral
and dorsal side. When the egg is fertilized, what will be the dorsal side of the embryo?
21. At a certain moment the follicle cells nearest to the nucleus
specialize as dorsal follicle cells. Which protein from the oocyte
causes this?
Parasegmentation
22. This is a Drosophila egg with in red the concentration of the
maternal gene product bicoid mRNA. Above is the graphical
representation of the left end of the egg, and the bicoid mRNA
concentration. Before fertilization there is a distinct area in the
egg where bicoid mRNA is found.
A. At which side will the head be formed?
B. After fertilization bicoid mRNA is translated into Bicoid protein, which diffuses to the posterior end. Why
is it not distributed evenly at the end of this stage?
o diffusion is too slow
o it has a short half-life
o inhibited transcription
C. Why does the bicoid mRNA itself not diffuse to the posterior end?
23. The Bicoid concentration activates gap genes in the zygote cells. These in their turn activate or inhibit the
pair-rule genes. Pair-rule genes express themselves in 7 bands across the zygote, thus dividing it into 14
parasegments.
A. It is not possible to find a common rule for all the 7 bands of one pair-rule gene to determine whether it
will express itself. So how are they regulated?
o by several more gap genes
o by several regulatory regions
o by inhibition from each other
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B. Consider the regulation of the third parasegment,
determined by the second stripe of Even-skipped.
What would be the regulatory pattern needed for this
band?
Bicoid:
Krüppel:
Hunchback:
Giant:
Segmentation
24. Expression of even-skipped (stained black) and fushi tarazu (stained
brown) at the end of stage 4. These activate the segment polarity
genes in stage 5.
This is the schematic representation of a small section of
the egg. Even-skipped and Fushi tarazu are dividing the egg
in 14 parasegments.
As you see the parasegments are only about 3 cells wide.
A. The pair-rule genes activate two segment polarity genes
which have key roles in segmentation. What are the
names of these genes?
B. In which cells do you expect engrailed to be expressed?
25. These cells were stained for Engrailed. Where will the segment
boundaries be formed?
26. In a series of blastoderm stage embryos, you injected one single cell in each embryo with a blue stain.
Shortly after, you fixed the embryos and counterstained with an antibody against Engrailed. Which of the
images below show(s) possible outcomes of the experiment?
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27. The parasegment boundary is established and maintained via intercellular signaling.
A. Make this statement correct:
. . . . . . . . stimulates/suppresses expression of . . . . . . . . . . in the adjacent/same
parasegment.
. . . . . . . . on its turn activates hedgehog expression, which stimulates/suppresses
expression of . . . . . . . ..
B. At later stages, the expression of these genes will become independent of each other. Why are
adjacent cells of the wingless expressing cells in the same segment not stimulated to express
engrailed?
28. The parasegment boundary is a boundary of cell lineage restriction. Cells and their descendants from one
parasegment never move into adjacent ones. These domains of lineage run through the segments. What is
the name for these domains?
Segment identity
29. So far the embryo has:
- a posterior, anterior, dorsal and ventral side (maternal genes)
- parasegmentation (gap and pair-rule genes)
- segmentation (segment polarity genes)
A. All segments are different. Which class of genes defines the segment identity?
B. What is a . . . . . . . . . . . . . gene?
o a gene that regulates another gene
o a gene that regulates many other genes
o a gene that codes for a transcription factor
o a gene that codes for a complex animal structure
C. What is the definition of a . . . . . . . . . . . . gene?
o a gene that regulates development of a whole structure
o a gene that when mutated results in homeosis
o a gene that only regulates other genes
o a gene that has analogues in vertebrate genes
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30. By the products of which genes is the expression of these genes controlled?
o Maternal genes
o Gap genes
o Pair-rule genes
o Segment polarity genes
31. Is the identity of all segments determined by these genes?
o Yes
o No. The most anterior segments of the head are determined otherwise
o No. The terminal segments are determined otherwise
o No. Only thorax and abdomen segments are determined this way
32. During the development of the fruit fly not all genes are constantly active. Indicate the period in which the
groups of genes are active:
gene group
oogenesis
first day
2-10 days
adult fly
33. Segment polarity genes and homeotic selector genes are activated by gap and/or pair-rule genes. These
proteins however are no longer active after about four hours. Segment polarity genes stimulate each other
so expression of them no longer needs to be stimulated by pair-rule genes after these have ceased their
activity.
The correct expression of selector homeotic genes is continued with the aid of the proteins of the Polycomb
and Trithorax gene groups. How do these continue the correct expression of homeotic selector genes?
o by stimulating their expression
o by maintaining their expression and repression
o by taking over their regulatory functions
34. There are two homeotic gene complexes in Drosophila. One complex of five homeobox genes controls the
behaviour of the parasegments 1-5.
A. How is this HOM complex called?
B. The other complex consists of three homeobox genes, and controls the behaviour of the parasegments
5-14. How is this HOM complex called?
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35. The three genes of the bithorax complex act in a combinational manner. In a
normal fruit fly the parasegments and segments develop as is shown right
(T=thorax, A=abdomen).
A. What will be the parasegment identity if the whole complex
is missing?
B. Ultrabithorax is one of the genes of the bithorax complex. It
controls the identity of parasegments 5 and 6. What will be
the parasegment identity if only this gene is missing?
C. What will be the parasegment identity when this is the only
gene present of the bithorax group?
D. Abdominal-B is another of the genes of the bithorax
complex. It controls the identity of parasegments 10 - 14.
What will be the parasegment identity when this gene is
missing?
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