Samenvatting van de hoorcolleges van Ontwikkelingsbiologie en Genetica (B-B3OBG05). De samenvatting is in het engels, aangezien de hoorcolleges ook in het engels waren.
HC 10, 11, 12, 13, 14, 15, 16 en 17
Ontwikkelingsbiologie en Genetica – HC’s deeltentamen
2
HC 10: Drosophila germline stem cells
Drosophila melanogaster
- Drosophila has a short life cycle: 10 days
- Easy to grow
- Extensive background in genetics, and a lot of tools
- Multicellular organism
- The molecular processes and pathways are conserved
Forward genetics = where you have a phenotype and you look for the genetic foundation of
this phenotype
- Mutant screens: chemical mutagenesis or transposon insertions
Reverse genetics = you have a gene and you look for its function
- Overexpression
- Transposon local hopping to create imprecise excissions: deletions
- RNAi
- Knockout by CRISPR Cas9
Stem cell regulation
1. External signals (extrinsic) = niche provides signal
2. Intrinsic signals = stem cell lineage. The cells just ‘know’ that they have to divide
asymmetrically
Germline stem cells are regulated by external signals
The abdomen is where the germline stemcells reside. They
divide, and one cell becomes a sperm or oocyt and the other
becomes a support cell that surround the germ cells.
The niche causes an asymmetric cell division, where both cells
have different cell fates.
At the tip of the abdomen are two ‘artichoke’ like structures.
Each structure has a string of beads with the development of
oocytes. The stem cells are at the very tip (on the left).
,Tip of the ovariole has 3 ‘stem cells’:
1. Germ line stem cell (GSC), gives rise to: cystoblasts
(CB)
2. Escort stem cells (ESC), gives rise to escort cells.
They escort the GSCs and provide certain factors
needed for the differentiation of the CB.
3. Follicle stem cells (FSC) give rise to follicle cells
(FC). They surround the egg chamber and they are
important in patterning the oocyte.
The Cap cells are the ‘niche’ for the GSC.
What signals are required to maintain GSCs?
Niche = specific location in a tissue where stem 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, the cell that stays behind is
still under the influence of the niche and stays a GSC.
Dpp (BMP/TGF-beta) is required for germline stem cell fate. It activates
Smads:
1. Dpp is a secreted ligand and it binds to two receptors (a receptor
heterodimer)
2. The receptor complex phosphorylates Smad
3. Smads can associate with other similar proteins (co-Smads) and
together they regulate transcription.
One of the target genes of Dpp is bam (bag of marbles), this gene is repressed
by Dpp. In a bam mutant you see the phenotype that there are only GSCs, no
egg chambers or cysts. Overexpression of Dpp has a similar phenotype as a
bam mutant.
The GSC is associated with a Cap cell and receives a Dpp signal from the Cap cells. The Smad
complex binds to a silencer complex of the bam gene and represses transcription. After
division, the two cells are pinched off (there is no connection anymore). The cystoblast is
pushed away and it is not under influence of the Cap cell anymore. It doesn’t receive Dpp
signaling anymore and the bam gene is on. This initiates cystoblast differentiation.
,Cap cells secrete Dpp, how is this regulated? Do the terminal filament
cells do that? Yes!
The terminal filament cells secrete the protein Unpaired. This is a
ligand of the JAK/STAT pathway: Janus Kinase-Signal Transducers and
Activators of Transcription.
Experiments to prove involvement in a process?
- Knockout unpaired
o In loss of function mutants you would expect no
germline stem cells and/or no Dpp pathway activation
- Overexpression experiment
o In gain of function mutants you would expect more
GSCs and maybe more Dpp pathway activation.
GAL-4/UAS = a genetic tool for overexpression experiments, where you can activate tissue
specific expression. You need to generate transgenic lines by P-element mediated
transformation.
- Ectopic expression of unpaired induces ectopic phosphorylated Smad in the ovary.
More Unpaired more Dpp
So:
1. The terminal filament cells produce Unpaired
2. Unpaired activates JAK/STAT signaling in Cap cells
3. Cap cells produce BMP ligands (Dpp)
4. Dpp signaling in GSC represses Bam and thus
differentiation
5. When the daughter cell moves away, the cell will
differentiate
Early oogenesis in the germarium
The egg chambers are generated in the germarium.
There are several regions in the germarium:
1. Region 1: asymmetric divisions of germline
stemcells
a. New germline stem cells
b. Cystoblasts: 4 rounds of incomplete
divisions to form 16 interconnected cells
2. Region 2: one cell differentiates as the oocyte in
the egg chamber, the others become nurse cells
3. Region 3: oocyte becomes polarized and nurse
cells start to polyploidize (doubling of the DNA,
but it’s not pulled apart).
, During the first incomplete division, cells are connected by ring canals and fusome plug
forms (these go through the holes). This way the cells become interconnected in the
Drosophila germline.
Fusomes = small membranous vesicles kept together by components of cytoskeleton. They
anchor one pole of the mitotic spindle.
During the next divisions, new fusome plugs will form which will fuse with the previous one.
You end up with 16 interconnected cells. One cell becomes the oocyte (oo). 15 cells are the
nurse cells (nc). Function: The nurse cells dump maternal products into the oocyte through
the ring canals. The embryo needs maternal products to fulfil the first divisions that happen
very rapidly when there is no zygotic transcription yet.
Microtubules are important for transport
- Microtubule motors ‘walk’ along the
microtubule cables
o Dynein moves towards the minus
end
o Kinesin moves towards the plus end
- Cargo is transported along microtubules,
such as maternal products.
Drosophila par-1 is required for localization of
posterior determinants (like oskar RNA).
Polarization by par-1 is in response to signaling from posterior follicle cells.
Drosophila spermatogenesis
- The GSC divides into another GSC and a gonialblast (Gb).
- 2 Cyst cells surround developing germline cells.
- After 4 transit amplifying (TA) divisions of the Gb they become 16 interconnected
spermatogonia.
Male: 16 interconnected spermatogonia.
Female: 16 interconnected cystocytes
Both have ring canals with fusomes that connect the cells
unpaired mRNA is expressed in the apical hub in the tip of
the testis. So the hub is the niche, and Unpaired is the niche
signal.
JAK/STAT pathway is activated in cells next to the hub, this
causes Stat expression (surrounding the hub).
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