Ontwikkelingsbiologie deel 2 Invertebrate model systems
HC 2.1 + 2.2 – Drosophila Melanogaster
The lifecycle
The lifecycle of a Drosophila is only 10 days long. Is starts with a fertilized egg that cleaves.
After cleaving a syncytial blastoderm will form. This will develop into a cellular blastoderm.
After gastrulation the embryo hatches and three larval stages follow. After the third larval
stage a pupa forms. The pupa goes into metamorphosis and an adult fly has developed.
Drosophila are easy to grow and there is much known about the genetics. Humans have only
twice as much genes as the Drosophila. 14.000 of their genes encode for proteins. The
molecular pathway and processes are conserved and the fly is a multicellular organism. This
makes it interesting to research the interaction between cells in the fly.
The early embryo is a syncytium. This means there are many nuclei present, but only one
cell membrane. After 90 minutes the nuclei migrate to the periphery of the cytoplasm. After
three hours, the nuclei have formed membranes and the embryo is now called a cellular
blastoderm. The syncytium is formed due to rapid mitosis with nuclear divisions only. Due to
these fast divisions, there is no time to produce cell membranes or proteins. The proteins
that enable division to occur are maternal and already present in the oocyte. The nuclei
‘know’ where to locate themselves due to the presence of a morphogen gradient. A
morphogen is a signalling molecule that contains positional information and can function as
a transcriptionfactor (Tcf).
During gastrulation the three germ layers are formed: (figure 3)
- Mesoderm migrates to internal locations
o This is due to twist expression in the mesoderm.
- Cells of the nervous system at the ventral side move inwards
- Endoderm and mesoderm move inside
- The foregut and hindgut grow inward
- The ectoderm remains outside.
During the gastrulation germband extension will occur as well. At first the yolk is covered by
a layer called amnioserosa: this is an extra-embryonic structure. It is a temporarily solution
to prevent leaking of the yolk. After a while dorsal closure will occur. A cell layer migrates
from ventral and close at the dorsal side, covering the entire embryo. This occurs after
retraction of the germband.
The embryo is segmented into 14 segments. 3 will form the mouth and head, 3 the
thorax and 8 the abdomen. On the ventral side of each segment, tooth-like outgrowths of
the epidermis are found, called the denticles. Imaginal discs are certain groups of epidermal
cells that are formed in the embryo and are present in the larva, that will develop into the
adult structures during metamorphosis.
Patterning of the embryo
Along the AP-axis the head, thorax and abdomen can be found. Along the DV-axis the
mesoderm, ventral, neuro and dorsal ectoderm and the amnioserosa can be found. The
main axes are already specified before fertilization in the oocyte. These are determined by
the maternal product and the maternal and zygotic gene patterns of the embryo.
1
, In the ovaries of a Drosophila the formation of a follicle cell starts in the so-called
germarium, with stem cells. In the germarium the stem cells divide asymmetrically. These
stem cells will divide into another stem cell and al clump of cells called the cystoblast. The
cystoblast divides into 16 interconnected cells. One of the 16 cells becomes the oocyte, the
others develop in nurse cells. Nurse cells provide the oocyte with maternal products (mRNAs
and proteins). All the 16 cells are surrounded by somatic follicle cells, which form the egg
chamber. Due to signalling the oocyte will move towards the posterior side of an older egg
chamber.
The nurse cells produce Delta that interacts with its receptor Notch, present on polar
follicle cells. These polar follicle cells express a ligand for the JAK/STAT pathway. Activation
of this pathway induces formation of a stalk by the follicle cells in between egg chambers.
The stalk cells upregulate cadherin and anchor younger oocytes at the posterior site. Gurken
mRNA is translated at the posterior site of the anchored oocyte and is secreted to the follicle
cells. Here, Gurken (=ligand) binds to the Torpedo receptor and thereby decides posterior
cell fate. The posterior follicle cells induce reorientation of the microtubule cytoskeleton in
the oocyte. → The oocyte moves to posterior as a result of signalling from the older egg
chamber. This is cell fate specification of follicle cells.
*Signalling between the oocyte and the follicle cells.
Nuclear migration is possible due to growing microtubules from the centrosome. These
tubules push the nucleus of the oocyte to the anterior corner. The anterior corner, where
the nucleus will be situated, will become the dorsal side of the future embryo. Migration due
to a microtubule network is called microtubule reorganization and is done by kinesin (walks
from – to +) and dynein (walks from + to -) motor proteins. These proteins are responsible
for the localization of maternal mRNAs possible as well.
One maternal mRNA that is located to the anterior site is bicoid. This mRNA thereby
patterns the anterior of the embryo. Oskar is a mRNA always located at the posterior site.
Nano is mRNA from nurse cells and also brought to posterior by motor proteins.
Specification of the DV-axis
The DV axis is set up by signals from posterior follicle cells. These cells signal the oocyte
nucleus to migrate from posterior towards the dorsal side, where it will stay. Gurken protein
also plays part in the DV-axis, because it induces the dorsal follicle cell fate.
*Gurken induces posterior and dorsal follicle cell fates.
Specific genes are expressed at the dorsal-anterior follicle cells. The follicle cells surround
the oocyte and the nurse cells. The follicle cells in different regions have a different cell fate
and determine the AP- and DV-axis.
Overview axis in Drosophila
- Antero-posterior axis: The oocyte and nucleus are located posterior. Gurken induces
the posterior follicle cells (Torpedo receptor). The posterior follicle cells reorganize
the cytoskeleton. Bicoid mRNA is located anterior and oskar and nanos mRNA are
located posterior.
- Dorso-ventral axis: The nucleus of the oocyte moves dorsally, due to binding of
Gurken to its Torpedo receptor. Gurken also induces dorsal follicle cells.
2
,Overall it is concluded that the AP-axis is set up by:
1. Maternal genes,
2. Gap genes
3. Pair rule genes
4. Segment polarity genes.
There is a sequential expression of genes:
- Maternal genes: product provided by the mother in the egg. The products diffuse in
the syncytium.
o Describes as: homozygous mothers do not show the phenotype, but the
offspring does. This is not entirely true, as the mother does have the same
microtubule, actine and mRNAs present in the offspring.
- Zygotic genes: expressed by the embryo
- Selector genes: all homeobox genes.
This temporal sequence expresses a certain hierarchy.
Mutants: genes are named after their mutant phenotype.
*Anterior of the embryo bicoid and hunckback are active. Posterior nanos and caudal and
at the terminal ends torso and trunk are active.
Bicoid is necessary for the formation of anterior structures. It is a morphogen and expressed
in a gradient. Anterior, the concentration is the highest. Bicoid contains a homeobox, that
functions as a transcription factor to activate zygotic genes. It can bind to RNA and has a
short half-life. Because of this high levels of bicoid are only present anterior: diffusion
towards posterior is not possible because bicoid is broken down before reaching the
posterior site. Bicoid protein is present in cell nuclei, and bicoid mRNA is present in the ECM.
In the unfertilized egg, bicoid mRNA is present anterior. Other maternal proteins
localize bicoid here. There is no Bicoid protein present yet, this is only present in nuclei after
fertilization.
Bicoid activates the maternal factor hunchback anterior. When Bicoid is present in
high levels, a rise in the concentration of Hunchback can be seen.
Caudal is a uniform maternal mRNA with a homeobox domain. A domain of bicoid
can bind to the mRNA of caudal and thereby repress the translation of caudal in the anterior
region of the embryo. This is done by preventing the binding of a cap-binding complex and
initiation of translation. The caudal gradient is at peak levels most posterior (where no bicoid
is present). Caudal plays part in the development of abdominal segments.
Hunchback is activated by bicoid and is a Zink finger. This transcription factor is
restricted to the anterior site
To conclude Bicoid functions as:
- Transcription factor: activating Hunchback anterior.
- RNA-binding protein: repressing Caudal translation anterior.
Nanos is an mRNA that is localized posteriorly. It is translated after fertilization and functions
as a Zink finger, that can bind RNA. By doing so, it supresses translation of Hunchback and
other mRNAs. Nanos does this with the help of Pumilio. Pumilio binds to a Nanos respons
3
, element (NRE) on the 3’-end of mRNA. Pumilio, Nanos and other proteins then form a
complex and inhibit translation.
- In the early embryo: Hunckback is inhibited
- In the pole and germ cells: several mRNAs are inhibited
- In the soma: several mRNAs are inhibited
There is translational repression of Hunckback by Nanos and Pumilio and repression of
Caudal by Bicoid.
HC 2.3 + 2.4 – Drosophila
Invertebrate model systems
There are three classes of maternal genes:
Anterior → bicoid, hunchback
Posterior → nanos, caudal
Terminal on both ends → torso, trunk
Torso specifies the terminal ends of the embryo, it is a receptor it is at on membrane which
is translated after fertilization. Torso is only activated at the poles but it is present over the
whole body. Trunk is the ligand for the Torso and is secreted into the perivitelline space,
which is between the embryo and vitelline membrane and Trunk is distributed uniformly. It
is an inactive ligand.
There are only effect of Trunk-Torso binding because of the follicle cells in the posterior
region. Torso-like is produced by these follicle cells at the poles and it and processes Trunk.
There is only procession of the inactive Trunk at the poles: this makes Trunk ligand active.
Processed trunk is in the perivitelline space and in small quantities at the poles. It doesn’t
fuse away because it is binded to the Torso receptor, and that is why signaling is only at the
poles. Trunk doesn’t move further than the receptors. Torso is a tyrosine kinase receptor.
→ Trunk binds to receptor Torso that leads to auto phosphorylation and proteins can bind to
this site and regulate the transcription of target genes.
4
HC 2.1 + 2.2 – Drosophila Melanogaster
The lifecycle
The lifecycle of a Drosophila is only 10 days long. Is starts with a fertilized egg that cleaves.
After cleaving a syncytial blastoderm will form. This will develop into a cellular blastoderm.
After gastrulation the embryo hatches and three larval stages follow. After the third larval
stage a pupa forms. The pupa goes into metamorphosis and an adult fly has developed.
Drosophila are easy to grow and there is much known about the genetics. Humans have only
twice as much genes as the Drosophila. 14.000 of their genes encode for proteins. The
molecular pathway and processes are conserved and the fly is a multicellular organism. This
makes it interesting to research the interaction between cells in the fly.
The early embryo is a syncytium. This means there are many nuclei present, but only one
cell membrane. After 90 minutes the nuclei migrate to the periphery of the cytoplasm. After
three hours, the nuclei have formed membranes and the embryo is now called a cellular
blastoderm. The syncytium is formed due to rapid mitosis with nuclear divisions only. Due to
these fast divisions, there is no time to produce cell membranes or proteins. The proteins
that enable division to occur are maternal and already present in the oocyte. The nuclei
‘know’ where to locate themselves due to the presence of a morphogen gradient. A
morphogen is a signalling molecule that contains positional information and can function as
a transcriptionfactor (Tcf).
During gastrulation the three germ layers are formed: (figure 3)
- Mesoderm migrates to internal locations
o This is due to twist expression in the mesoderm.
- Cells of the nervous system at the ventral side move inwards
- Endoderm and mesoderm move inside
- The foregut and hindgut grow inward
- The ectoderm remains outside.
During the gastrulation germband extension will occur as well. At first the yolk is covered by
a layer called amnioserosa: this is an extra-embryonic structure. It is a temporarily solution
to prevent leaking of the yolk. After a while dorsal closure will occur. A cell layer migrates
from ventral and close at the dorsal side, covering the entire embryo. This occurs after
retraction of the germband.
The embryo is segmented into 14 segments. 3 will form the mouth and head, 3 the
thorax and 8 the abdomen. On the ventral side of each segment, tooth-like outgrowths of
the epidermis are found, called the denticles. Imaginal discs are certain groups of epidermal
cells that are formed in the embryo and are present in the larva, that will develop into the
adult structures during metamorphosis.
Patterning of the embryo
Along the AP-axis the head, thorax and abdomen can be found. Along the DV-axis the
mesoderm, ventral, neuro and dorsal ectoderm and the amnioserosa can be found. The
main axes are already specified before fertilization in the oocyte. These are determined by
the maternal product and the maternal and zygotic gene patterns of the embryo.
1
, In the ovaries of a Drosophila the formation of a follicle cell starts in the so-called
germarium, with stem cells. In the germarium the stem cells divide asymmetrically. These
stem cells will divide into another stem cell and al clump of cells called the cystoblast. The
cystoblast divides into 16 interconnected cells. One of the 16 cells becomes the oocyte, the
others develop in nurse cells. Nurse cells provide the oocyte with maternal products (mRNAs
and proteins). All the 16 cells are surrounded by somatic follicle cells, which form the egg
chamber. Due to signalling the oocyte will move towards the posterior side of an older egg
chamber.
The nurse cells produce Delta that interacts with its receptor Notch, present on polar
follicle cells. These polar follicle cells express a ligand for the JAK/STAT pathway. Activation
of this pathway induces formation of a stalk by the follicle cells in between egg chambers.
The stalk cells upregulate cadherin and anchor younger oocytes at the posterior site. Gurken
mRNA is translated at the posterior site of the anchored oocyte and is secreted to the follicle
cells. Here, Gurken (=ligand) binds to the Torpedo receptor and thereby decides posterior
cell fate. The posterior follicle cells induce reorientation of the microtubule cytoskeleton in
the oocyte. → The oocyte moves to posterior as a result of signalling from the older egg
chamber. This is cell fate specification of follicle cells.
*Signalling between the oocyte and the follicle cells.
Nuclear migration is possible due to growing microtubules from the centrosome. These
tubules push the nucleus of the oocyte to the anterior corner. The anterior corner, where
the nucleus will be situated, will become the dorsal side of the future embryo. Migration due
to a microtubule network is called microtubule reorganization and is done by kinesin (walks
from – to +) and dynein (walks from + to -) motor proteins. These proteins are responsible
for the localization of maternal mRNAs possible as well.
One maternal mRNA that is located to the anterior site is bicoid. This mRNA thereby
patterns the anterior of the embryo. Oskar is a mRNA always located at the posterior site.
Nano is mRNA from nurse cells and also brought to posterior by motor proteins.
Specification of the DV-axis
The DV axis is set up by signals from posterior follicle cells. These cells signal the oocyte
nucleus to migrate from posterior towards the dorsal side, where it will stay. Gurken protein
also plays part in the DV-axis, because it induces the dorsal follicle cell fate.
*Gurken induces posterior and dorsal follicle cell fates.
Specific genes are expressed at the dorsal-anterior follicle cells. The follicle cells surround
the oocyte and the nurse cells. The follicle cells in different regions have a different cell fate
and determine the AP- and DV-axis.
Overview axis in Drosophila
- Antero-posterior axis: The oocyte and nucleus are located posterior. Gurken induces
the posterior follicle cells (Torpedo receptor). The posterior follicle cells reorganize
the cytoskeleton. Bicoid mRNA is located anterior and oskar and nanos mRNA are
located posterior.
- Dorso-ventral axis: The nucleus of the oocyte moves dorsally, due to binding of
Gurken to its Torpedo receptor. Gurken also induces dorsal follicle cells.
2
,Overall it is concluded that the AP-axis is set up by:
1. Maternal genes,
2. Gap genes
3. Pair rule genes
4. Segment polarity genes.
There is a sequential expression of genes:
- Maternal genes: product provided by the mother in the egg. The products diffuse in
the syncytium.
o Describes as: homozygous mothers do not show the phenotype, but the
offspring does. This is not entirely true, as the mother does have the same
microtubule, actine and mRNAs present in the offspring.
- Zygotic genes: expressed by the embryo
- Selector genes: all homeobox genes.
This temporal sequence expresses a certain hierarchy.
Mutants: genes are named after their mutant phenotype.
*Anterior of the embryo bicoid and hunckback are active. Posterior nanos and caudal and
at the terminal ends torso and trunk are active.
Bicoid is necessary for the formation of anterior structures. It is a morphogen and expressed
in a gradient. Anterior, the concentration is the highest. Bicoid contains a homeobox, that
functions as a transcription factor to activate zygotic genes. It can bind to RNA and has a
short half-life. Because of this high levels of bicoid are only present anterior: diffusion
towards posterior is not possible because bicoid is broken down before reaching the
posterior site. Bicoid protein is present in cell nuclei, and bicoid mRNA is present in the ECM.
In the unfertilized egg, bicoid mRNA is present anterior. Other maternal proteins
localize bicoid here. There is no Bicoid protein present yet, this is only present in nuclei after
fertilization.
Bicoid activates the maternal factor hunchback anterior. When Bicoid is present in
high levels, a rise in the concentration of Hunchback can be seen.
Caudal is a uniform maternal mRNA with a homeobox domain. A domain of bicoid
can bind to the mRNA of caudal and thereby repress the translation of caudal in the anterior
region of the embryo. This is done by preventing the binding of a cap-binding complex and
initiation of translation. The caudal gradient is at peak levels most posterior (where no bicoid
is present). Caudal plays part in the development of abdominal segments.
Hunchback is activated by bicoid and is a Zink finger. This transcription factor is
restricted to the anterior site
To conclude Bicoid functions as:
- Transcription factor: activating Hunchback anterior.
- RNA-binding protein: repressing Caudal translation anterior.
Nanos is an mRNA that is localized posteriorly. It is translated after fertilization and functions
as a Zink finger, that can bind RNA. By doing so, it supresses translation of Hunchback and
other mRNAs. Nanos does this with the help of Pumilio. Pumilio binds to a Nanos respons
3
, element (NRE) on the 3’-end of mRNA. Pumilio, Nanos and other proteins then form a
complex and inhibit translation.
- In the early embryo: Hunckback is inhibited
- In the pole and germ cells: several mRNAs are inhibited
- In the soma: several mRNAs are inhibited
There is translational repression of Hunckback by Nanos and Pumilio and repression of
Caudal by Bicoid.
HC 2.3 + 2.4 – Drosophila
Invertebrate model systems
There are three classes of maternal genes:
Anterior → bicoid, hunchback
Posterior → nanos, caudal
Terminal on both ends → torso, trunk
Torso specifies the terminal ends of the embryo, it is a receptor it is at on membrane which
is translated after fertilization. Torso is only activated at the poles but it is present over the
whole body. Trunk is the ligand for the Torso and is secreted into the perivitelline space,
which is between the embryo and vitelline membrane and Trunk is distributed uniformly. It
is an inactive ligand.
There are only effect of Trunk-Torso binding because of the follicle cells in the posterior
region. Torso-like is produced by these follicle cells at the poles and it and processes Trunk.
There is only procession of the inactive Trunk at the poles: this makes Trunk ligand active.
Processed trunk is in the perivitelline space and in small quantities at the poles. It doesn’t
fuse away because it is binded to the Torso receptor, and that is why signaling is only at the
poles. Trunk doesn’t move further than the receptors. Torso is a tyrosine kinase receptor.
→ Trunk binds to receptor Torso that leads to auto phosphorylation and proteins can bind to
this site and regulate the transcription of target genes.
4
