Hoorcollege 10: Drosophilia germline stem cells
Drospohilia melanogaster
- has a short life cycle (10 days)
- is very easy to grow
- has an extensive background in genetics
- is a multicellular organism
- has very conserved molecular processes and pathways
Genetics
Forward genetics
- Mutant screens
o Chemical mutagenesis
o Transposon insertions
Reverse genetics
- Overexpression
- Transposon local hopping to create imprecise excisions: deletions
- RNAi: expression of hairpin transgenes
- “knock out” by CRISPR/Cas9
There are many genetic tools:
- Markers, balancers
- Expression of genes in specific tissues and at different times
- Clonal analysis
Drosophila as a genetic system: each tissue can be a model system for different processes
Stem cells
External signals: niche provides signals / intrinsic signals: stem cell lineage
Drosophilia males can be recognized by the testis located at the tip of the abdomen, dark
dome like structure. Females lack this and have a vaginal plate.
- Abdomen is also where the germline stem cells lay. Stem cells can become germ cells
or support cells that surround the germ cells.
- Stem cells undergo asymmetric division
- Signaling from the niche
- One cell will have a different cell fate than another
,Female germline
In the ovaries, one cell will differentiate into an oocyte when it comes near the exit
- One unit is called a germarium, the more it progresses it will be called an egg
chamber.
- Stem cells locate at the tip (germarium side). You could prove this by preforming a
transplant of the tip of the ovarioles into a different female. It will give rise to eggs
and adult flies. You could also preform an ablation experiment in which you remove
it.
There are 3 stem cells in the Drosophila ovariole:
- Germ line stem cell (GSC) will give rise to eventually
oocyte and another stem cell, cystoblast (CB)
- Escort stem cells (ESC) will give rise to escort cells
that escort stem cells and will provide factors that are
needed for the differentiation of the cystoblasts
- Follicle stem cells (FSC) will give rise to follicle cells
(FC) which surround an egg chamber and are need for
pattering the oocyte.
The Cap cells are the niche for the GSC
What signals are required to maintain GSCs?
- Notch? Wnt? BMPs?
Stem cell niche GSC:
- Niche: specific location in a tissue where sperm cells can reside for an indefinite
period of time and produce progeny cells while self-renewing
- A small number of stem cells are anchored to their stromal partner cells that produce
signals
- The niche produces micro environmental factors
- Oriented cell divisions direct stem cell daughters away from the stem cell
microenvironment or niche
In dpp temperature sensitive mutants they have found that
the mutant becomes sterile. The germline marker will
disappear in the niche.
➔ Dpp is a BMP and it is required for the germline
stem cell fate.
,What is dpp?
Dpp/TGFbeta/BMP activate Smads. It is a secreted ligand and it binds to a receptor
heterodimer. The receptor complex phosphorylates Smad. Smad can associate itself
with CoSmads en together they translocate to the nucleus and regulate transcription of
Dpp target genes.
➔ Phosphorylated Smads are markers for dpp signaling.
One of the target genes of dpp is bag-of-marbles (bam). In the wildtype bam-GFP is
on in all the stem cells, when these cells want to differentiate, they need bam
expression. In a bam mutant with bam-GFP you still see small green cells because GFP
is expressed under influence of the bam promoter, there is no differentiation into
cystoblasts. You will only see GSC (that are not able to differentiate) and no cysts or
egg chambers. Overexpression of Dpp has similar phenotype as bam mutant because
overexpression causes a negative feedback to bam. In overexpression this causes a repression
of bam this will also cause a loss of oocytes because they can’t differentiate.
➔ Dpp suppresses bam
GSC is associated with Cap cell and receives Dpp signal from this Cap cell and it represses
bam. Smad complex translocates to the nucleus of GSC and
interconnected daughter. Smad complex binds to silencer
complex of bam gene and represses transcription. After division
the two cells will pinch off. No connection between GSC and
cystoblast. The cystoblast is now pushed away. It is out of the
influence of the Cap cell and does not receive Dpp signaling
anymore which causes bam transcription to be turned on.
➔ When cytokinesis is completed bam becomes de-
repressed and initiate cystoblast differentiation
How is Dpp/BMP expression regulated?
Dpp/BMP is produced by the Cap cells. Cap cells are next to terminal filament cells.
➔ Do terminal filament cells regulate Dpp expression?
Unpaired is a secreted ligand secreted by the terminal filament cells. Unpaired is a ligand of
the JAK/STAT pathway.
JAK/STAT: Janus Kinase-Signal Transducers and Activators of Transcription. It is conserved
in Drosophila, except for the ligand.
- Ligand is Unpaired (Udp), not a cytokine as in mammals
- Receptor is Domeless (DOME)
- Kinase associated with receptor is JAK:Hopscotch (HOP)
- Transcription factor is STAT:STAT92E
Which experiments can be done to prove involvement in a process?
- Loss of function mutants
o You expect there to be no germline stem cells and no Dpp
pathway activation.
- Gain of function mutants or ectopic expression
o You expect there to be more germline stem cells and more or
ectopic Dpp pathway activation.
, Ectopic expression can be done with the use of genetic tools
- Generate transgenic line by P-element mediated transformation
- Tissue specific expression by GAL-4/UAS
Ectopic expression of unpaired induces ectopic phosphorylated Smad in the ovary.
➔ Dpp signaling
Signaling in the female GSC niche: Unpaired produced from the Terminal filament cells
activate JAK/STAT signaling in Cap Cells. Cap cells will produce BMP ligands. Dpp
signaling in GSC represses bam and differentiation
Early oogenesis in the germarium
- Egg chambers are generated in the germarium
- Region 1: asymmetric divisions of germ line stem cell
o New germline stem cell
o Cystoblast:
▪ 4 rounds of incomplete divisions to form 16 interconnected cells
▪ Cystocystes
- Region 2: one cell differentiates as the oocyte in the egg chamber, the others become
nurse cells
- Region 3: oocyte becomes polarized and nurse cells start to polyploidize (doubles
DNA but does not pull them apart)
Interconnected cells in the Drosophila germline
- During the first incomplete division cells are connected by
ring canals and a fusome plug forms
- Fusome
o Small membranous vesicles kept together by
components of cytoskeleton
o Anchor one pole of the mitotic spindle
- During the next divisions new fusome plugs will form which
will fuse with the previous one
- One cell will become the oocyte and 15 cells will be nurse
cells, they will dump maternal product into the oocyte and the
nurse cells degenerate. The maternal products pass through
the ring canal. Maternal products are needed for development,
because it does not have any cytotic transcription yet.
Microtubules are bundles that run through the ring canal. Microtubule motors ‘walk’ along
microtubule cables
- Dynein moves towards the minus end
- Kinesin moves towards the plus end
Cargo is transported along the microtubules. Maternal mRNA localization by microtubules
What determines Anterior/Posterior polarity in the oocyte?
Drosophila par-1 is required for localization of posterior determinants. Polarization by par-1
is in response to signaling from posterior follicle cells.
Drospohilia melanogaster
- has a short life cycle (10 days)
- is very easy to grow
- has an extensive background in genetics
- is a multicellular organism
- has very conserved molecular processes and pathways
Genetics
Forward genetics
- Mutant screens
o Chemical mutagenesis
o Transposon insertions
Reverse genetics
- Overexpression
- Transposon local hopping to create imprecise excisions: deletions
- RNAi: expression of hairpin transgenes
- “knock out” by CRISPR/Cas9
There are many genetic tools:
- Markers, balancers
- Expression of genes in specific tissues and at different times
- Clonal analysis
Drosophila as a genetic system: each tissue can be a model system for different processes
Stem cells
External signals: niche provides signals / intrinsic signals: stem cell lineage
Drosophilia males can be recognized by the testis located at the tip of the abdomen, dark
dome like structure. Females lack this and have a vaginal plate.
- Abdomen is also where the germline stem cells lay. Stem cells can become germ cells
or support cells that surround the germ cells.
- Stem cells undergo asymmetric division
- Signaling from the niche
- One cell will have a different cell fate than another
,Female germline
In the ovaries, one cell will differentiate into an oocyte when it comes near the exit
- One unit is called a germarium, the more it progresses it will be called an egg
chamber.
- Stem cells locate at the tip (germarium side). You could prove this by preforming a
transplant of the tip of the ovarioles into a different female. It will give rise to eggs
and adult flies. You could also preform an ablation experiment in which you remove
it.
There are 3 stem cells in the Drosophila ovariole:
- Germ line stem cell (GSC) will give rise to eventually
oocyte and another stem cell, cystoblast (CB)
- Escort stem cells (ESC) will give rise to escort cells
that escort stem cells and will provide factors that are
needed for the differentiation of the cystoblasts
- Follicle stem cells (FSC) will give rise to follicle cells
(FC) which surround an egg chamber and are need for
pattering the oocyte.
The Cap cells are the niche for the GSC
What signals are required to maintain GSCs?
- Notch? Wnt? BMPs?
Stem cell niche GSC:
- Niche: specific location in a tissue where sperm cells can reside for an indefinite
period of time and produce progeny cells while self-renewing
- A small number of stem cells are anchored to their stromal partner cells that produce
signals
- The niche produces micro environmental factors
- Oriented cell divisions direct stem cell daughters away from the stem cell
microenvironment or niche
In dpp temperature sensitive mutants they have found that
the mutant becomes sterile. The germline marker will
disappear in the niche.
➔ Dpp is a BMP and it is required for the germline
stem cell fate.
,What is dpp?
Dpp/TGFbeta/BMP activate Smads. It is a secreted ligand and it binds to a receptor
heterodimer. The receptor complex phosphorylates Smad. Smad can associate itself
with CoSmads en together they translocate to the nucleus and regulate transcription of
Dpp target genes.
➔ Phosphorylated Smads are markers for dpp signaling.
One of the target genes of dpp is bag-of-marbles (bam). In the wildtype bam-GFP is
on in all the stem cells, when these cells want to differentiate, they need bam
expression. In a bam mutant with bam-GFP you still see small green cells because GFP
is expressed under influence of the bam promoter, there is no differentiation into
cystoblasts. You will only see GSC (that are not able to differentiate) and no cysts or
egg chambers. Overexpression of Dpp has similar phenotype as bam mutant because
overexpression causes a negative feedback to bam. In overexpression this causes a repression
of bam this will also cause a loss of oocytes because they can’t differentiate.
➔ Dpp suppresses bam
GSC is associated with Cap cell and receives Dpp signal from this Cap cell and it represses
bam. Smad complex translocates to the nucleus of GSC and
interconnected daughter. Smad complex binds to silencer
complex of bam gene and represses transcription. After division
the two cells will pinch off. No connection between GSC and
cystoblast. The cystoblast is now pushed away. It is out of the
influence of the Cap cell and does not receive Dpp signaling
anymore which causes bam transcription to be turned on.
➔ When cytokinesis is completed bam becomes de-
repressed and initiate cystoblast differentiation
How is Dpp/BMP expression regulated?
Dpp/BMP is produced by the Cap cells. Cap cells are next to terminal filament cells.
➔ Do terminal filament cells regulate Dpp expression?
Unpaired is a secreted ligand secreted by the terminal filament cells. Unpaired is a ligand of
the JAK/STAT pathway.
JAK/STAT: Janus Kinase-Signal Transducers and Activators of Transcription. It is conserved
in Drosophila, except for the ligand.
- Ligand is Unpaired (Udp), not a cytokine as in mammals
- Receptor is Domeless (DOME)
- Kinase associated with receptor is JAK:Hopscotch (HOP)
- Transcription factor is STAT:STAT92E
Which experiments can be done to prove involvement in a process?
- Loss of function mutants
o You expect there to be no germline stem cells and no Dpp
pathway activation.
- Gain of function mutants or ectopic expression
o You expect there to be more germline stem cells and more or
ectopic Dpp pathway activation.
, Ectopic expression can be done with the use of genetic tools
- Generate transgenic line by P-element mediated transformation
- Tissue specific expression by GAL-4/UAS
Ectopic expression of unpaired induces ectopic phosphorylated Smad in the ovary.
➔ Dpp signaling
Signaling in the female GSC niche: Unpaired produced from the Terminal filament cells
activate JAK/STAT signaling in Cap Cells. Cap cells will produce BMP ligands. Dpp
signaling in GSC represses bam and differentiation
Early oogenesis in the germarium
- Egg chambers are generated in the germarium
- Region 1: asymmetric divisions of germ line stem cell
o New germline stem cell
o Cystoblast:
▪ 4 rounds of incomplete divisions to form 16 interconnected cells
▪ Cystocystes
- Region 2: one cell differentiates as the oocyte in the egg chamber, the others become
nurse cells
- Region 3: oocyte becomes polarized and nurse cells start to polyploidize (doubles
DNA but does not pull them apart)
Interconnected cells in the Drosophila germline
- During the first incomplete division cells are connected by
ring canals and a fusome plug forms
- Fusome
o Small membranous vesicles kept together by
components of cytoskeleton
o Anchor one pole of the mitotic spindle
- During the next divisions new fusome plugs will form which
will fuse with the previous one
- One cell will become the oocyte and 15 cells will be nurse
cells, they will dump maternal product into the oocyte and the
nurse cells degenerate. The maternal products pass through
the ring canal. Maternal products are needed for development,
because it does not have any cytotic transcription yet.
Microtubules are bundles that run through the ring canal. Microtubule motors ‘walk’ along
microtubule cables
- Dynein moves towards the minus end
- Kinesin moves towards the plus end
Cargo is transported along the microtubules. Maternal mRNA localization by microtubules
What determines Anterior/Posterior polarity in the oocyte?
Drosophila par-1 is required for localization of posterior determinants. Polarization by par-1
is in response to signaling from posterior follicle cells.
