Traﬃcking during asymmetric cell division in zebraﬁsh
Daniel Abegg — Pedro Surriabre
firstname.lastname@example.org — email@example.com
Assistants: Irinka Castanon — Claudia Campos
Universit´ de Gen`ve, Science II March 25, 2009
Introduction which then translocate to the nucleus and reg-
ulate the transcription of its target genes with
The zebraﬁsh, whose binomial name is Danio CSL
Renio, is an aquarium ﬁsh used frequently in The function of the mind bomb (mib) protein
the laboratory as an animal model in the ge- is to ubiquitin Delta which will therefore be-
netic researches. Its main characteristics are gin the internalization of Delta-Notch and this
its rapid embryonic development that takes facilitates the S2 cleavage of the extracellular
3 days from egg to larvae and the fact that part of Notch and the release of the NICD af-
its embryos can develop outside the mother, ter S3 (done by γ-secretase).
which helps the manipulation and observation.
A frequent technique used in research with this The photoconversion is a technique that allows
animal model is the usage of Morpholino. changing the color emitted by a ﬂuorescence
The morpholino is an oligonucleotide (usually protein permanently. This is helpful for track-
25 bases) that binds the RNA and blocks the ing the developing of a single cell. Usually the
access to another molecules. By doing this, ﬂuorescence protein used in this technique is
the morpholino prevent the translation of the Kaede, which is a cytosolic protein and thus
RNA into a protein. allows the observation of the whole cell.
The key point of the embryonic developing is The goal of this TP is to study the roles of Sara
the asymmetrical cellular division. During this endosomes and Notch signaling in the cell dif-
process, the Sara endosomes are distributed in ferentiation during the developing of the spinal
diﬀerent amounts along the two daughter cells. cord in the embryos of the zebraﬁsh. To study
This asymmetry of Sara endosomes segregation this, we will inject Sara1 morpholino in the
allows the diﬀerentiation of the cells. The Sara eggs of the zebraﬁsh in order to knock out the
endosomes are a subset of the early endosomes. activity of the Sara endosomes, and we will
In the same way the Notch signaling pathway study the phenotype of this mutant and also
has an important role in the diﬀerentiation the phenotype of the Mib (mind bomb) mu-
of the daughter cells by controlling the gene tant. We will perform a photoconversion using
expression. Indeed, after interaction between the protein Kaede. The RNA coding for Kaede
the receptor Notch and its ligand Delta, there will be injected as well as the RNA coding for
are two cleavages (S2 and S3) that release Sara-Venus into the embryos of zebraﬁsh.
the intracellular domain of Notch (NICD),
2 Traﬃcking during asymmetric cell division in zebraﬁsh
Methodology after asymmetrical division. Three days after
the cell photocoverted is imaged with a micro-
Subcellular localization of Sara endosomes scope.
First of all, 3 cocktails of RNA are prepared in
order to track the localizations of Sara endo- Results
somes and Rab proteins in the cells. The ﬁrst
cocktail contains Rab5-CFP, Sara-Venus and Hu Staining
GAP43-mRFP. The second cocktail contains
Rab11-CFP, Sara-Venus and GAP43-mRFP. The Hu staining results (ﬁgure 1) show diﬀer-
The third cocktail contains Rab7-CFP, Sara- ent phenotypes. The spinal cord of the mib
Venus and GAP43-mRFP. Then, these cock- mutation have an enormous amount of neurons
tails are injected into the eggs of zebraﬁsh; and but they are smaller than those of the control.
after that, the RNA’s non-introduced are re- The total number of cells in the center of the
covered by performing a precipitation of the spinal cord is smaller (seen on the DAPI pic-
RNA with LiCl. Finally, the imaging of these ture). The SARA morpholino display a little
embryos is done with a ﬂuorescence micro- more neurons (24 to 16 for the control) and the
scope. morphology is about the same.
Analysis of Sara morphants and Mib mu- Subcellular localization of SARA-positive
tants in the developing spinal cord endosomes
In the ﬁrst place, we generated the Sara mor- In this experiment we looked at the localization
phants embryos. In order to do so an injection of the endosomes with the SARA protein (ﬁg-
of the Sara1-splicing Morpholino is performed ure 2). It is seen that in the case of rab5 there
over the eggs of the zebraﬁsh. We also used is a massive colocalization of SARA1 with the
embryos containing a mutation over the Mib early endosomes. In the case of the late en-
protein. After that, the staining of the HU dosomes (rab7) there is only little colocaliza-
protein is done over the mutant embryos (Sara tion and ﬁnally with the recycling endosomes
morphants and Mib mutants) and over the con- (rab11) there is almost no colocalization.
trol embryos with primary and secondary an-
tibodies. The nuclei are colored with Dapi.
After that, the deyolking is done over the em-
bryos in order to recover the spinal cord of the
In a ﬁrst part it is seen that SARA endosomes
three types of embryos. The spinal cords are
is located at the separation zone which is in the
placed over a slide and the imaging is done with
middle of the cell (ﬁgure 3A). Then the ﬁgure
a ﬂuorescence microscope.
3B shows that the division of SARA is asym-
metric. This fact is interesting because it will
Photoconversion be used to determined the destiny of the two
First of all the embryos are injected with the The photoconvertible protein Kaede was used.
RNA coding for Sara1-CFP and with the RNA We choose to convert the cell with less SARA
coding for Kaede protein. After the develop- (on the left in ﬁgure 4B) after we quantiﬁed
ing of the embryos, the photoconversion is per- that amount per cell (ﬁgure 4A). The result is
formed over a cell containing a little bit of Sara that after three days which is plenty of time for
Traﬃcking during asymmetric cell division in zebraﬁsh 3
Figure 1: Hu staining results for the control (WT), the mib mutant and the sara-morpholino.
A. the staining pictures from the microscope. Dapi is in blue and Hu positifs neurons in red.
The withe lines are delimiting the counting of the neurons for the hitsogram. B. the histogram
of the number of neurons between three somites.
the cell to divide, there is still only one red cell tivate notch in a neighboring cell which will
present and therefore there was no division. cause proliferation. Lateral inhibition also
prevent the precursor pool from depletion
by the formation of lot of early born neuron
Discussion which seems to have a smaller size (ﬁgure 1
The function of the mind bomb (mib) protein The absence of SARA doesn’t seem to have
is to facilitates the S2 cleavage of the extracel- a strong eﬀect on cell diﬀerentiation because
lular part of Notch with an monoubiquitina- there was hardly more neurons compare to the
tion of Delta that will internalize with Notch- wild type (ﬁgure 1). This was quite a strange
extracellular domain and therefore allow (af- observation because in the photoconversion ex-
ter S3 cleavage) NICD to go activate target periment the cell which gets less sara becomes
genes. In the case of the mib mutant there is a neuron and here we have no sara at all and
the interaction between Delta and notch but the number of neuron isn’t much higher than
there is no internalization and therefore notch the control. We were awaiting a phenotype
is not cleaved and activated. This results in similar to the mib mutant but is seems that
a much higher number of neurons like shown the loss of sara is compensated by other pro-
on ﬁgure 1. This phenotype is like awaited teins and mechanisms.
because the activation of notch promotes pro- After the photoconversion the chosen cell
liferation of the cell . Normally there is a didn’t divide anymore even after three days
competition between the cell for which one which clearly show that it had become a neu-
will become a neuron (lateral inhibition) and ron. This means the daughter cell with less
this is promoted by the ligand Dalta. Cells sara will diﬀerentiate in opposition to the
therefore increase the concentration of Delta daughter with more sara who continue to pro-
at their surface to raise the probability to ac- liferate. This result indicate that sara plays
4 Traﬃcking during asymmetric cell division in zebraﬁsh
Figure 2: The diﬀerent Rabs are shown in line with rab5 on the early endosomes, then rab7 on
the late endosomes and rab11 on recycling endosomes. For each one there is the protein SARA
and then the merge with also a zoom. The white arrows shows the colocalisation (yellow dots)
of rab and SARA.
Figure 3: In green is SARA1-Venus and in red is palmitoylated mRFP. A. the position of SARA
in the central spindle area during mitosis of a 18 somite embryo. B. time frame pictures of the
asymmetric division of SARA in the two daughter cells.
an important role in the destiny of the two The observed colocalization of sara protein
daughter cells. Those observations put to- with rab7 and rab11 is not normal because they
gether would state that sara helps with the are only present on the early endosomes with
diﬀerentiation but is not the most important rab5. It could be that the multi-vesicular
player. body (MVB) contains some sara and it there-
fore is present on late endosomes and recycling
Traﬃcking during asymmetric cell division in zebraﬁsh 5
Figure 4: A. the asymmetric proportion of SARA-CFP in the two daughter cells (3x less for
one cell) and the cell witch will be photoconverted (PC). B. the photoconversion (from green
to red) and the results three days after the photoconversion. The white ellipse represents the
border of the cells
endosomes. Indeed sara has been obsered on sara in the daughters cells inﬂuence the des-
the MVB membrane. Therefore the concen- tiny of those cells. It would be interesting to
tration should be low like our observation in know the minimum amout of Sara that has to
ﬁgure 2. be inherited by one daughter cell to undergo
a proliferation instead of a diﬀeratiation into
two neurons. The second hypothesis propose a
bigger number of proliferative cells. This has
To explain the observed phenotypes there were not be observed directly in the experiment,
two predominant hypothesis. The ﬁrst one but it could be possible if there is occasionally
state that the higher number of neurons is a symmetrical division of the stem cell giving
caused by the diﬀerentiation of the precursor two proliferative cells instead of a asymmetri-
cell into two neurons, in this case the division cal division giving a neuron and a proliferative
would be symmetric. The other hypothesis to cell. In this case the pool of stem cell increase
explain the bigger observed number of neurons gradually and thus, in the end, the number of
would be that there were a lot more of precur- neurons increase too, and we would observe
sor cells that would divide asymmetrically into the same phenotype.
a proliferative cell and a neuron. The ﬁrst hy-
pothesis seems to apply better in the context
of the mib mutant, because if the cell divide
into two neurons, the precursor pool would The Delta-Notch interaction seem to be the
empty itself very fast and the total number major signaling pathway for the diﬀerentiation
of cells would be lower compared to the wild of the two daughter cells. This conclusion is
type. This diminution of cell is more or less based on the fact that the mib mutant, which
observed with the dapi staining in ﬁgure 1. directly interferes with this pathway, shows a
To check this hypothesis it would be neces- much greater variation in the number of neu-
sary to determined if the division is indeed ron compared to the wild type and to the sara
symmetric. It was said that the quantity of morphant. It is not known if sara interacts or
6 Traﬃcking during asymmetric cell division in zebraﬁsh
is somehow linked to the delta-notch pathway. References
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”J’atteste que dans ce texte toute aﬃrmation
qui n’est pas le fruit de ma r´ﬂexion person-
nelle est attribu´e ` sa source et que tout pas-
sage recopi´ d’une autre source est en outre
plac´ entre guillemets.”
Daniel Abegg Pedro Surriabre