1summer, 2011 ● national institute for basic biology

NIBB INTERNSHIP REPORTDistribution of diploids and tetraploids in a tetraploid↔diploid aggregation
chimera of fluorescently labeled Mus musculus embryos
Nan Pang, Summer 2011
Fujimori Laboratory, National Institute for Basic Biology
Myodaiji, Okazaki, Aichi 444-8585, JapanINTRODUCTION
Polyploidy indicates a cell that contains more than
two haploid (n) sets of chromosomes (Figure 1). It is
commonly observed among plants and might also beobserved in some “lower” animals; however, it is lessfrequently seen in most of the animals because of theircomplexity. For instance, polyploidy is rare in humans,
yet it is observed in human skeletal muscle cells.
Polyploid mammals can be experimentally produced
in a laboratory environment. Polyploid embryos can beused to provide evidence for further investigations ofcell size regulation, cell number, and rate of cell cleavage. Tetraploidy is more widely used than other forms
polyploidy in various studies. Although tetraploid–diploid (4n:2n) chimeras often survive the gestation period (Eakin and Behringer, 2003), tetraploid cells areunder-represented in the embryo proper or the innercell mass (ICM) because of selective disadvantageof diploid cells (Tarkowski et al., 1977). However, themechanism of under-representation and restriction oftetraploid cells remains unclear.
Conventionally, chimeras can be created by two
different techniques: blastocyst injection and aggregation. Each method has its advantages and disad- FIGURE 1. HAPLOID, DIPLOID, AND POLYPLOIDS
vantages, and methods may vary depending on the Visualizing the polyploidy. Triploids and tetraploids are considered asprocedure that is followed; however, the aggregation the polyploids; yet, tetraploidy is much rare than triploidy.
SOURCE: WIKIPEDIA COMMONS
method is preferred for tetraploid chimera production
in research because of its capability of mass production and ease (Wood et al., 1993). Tarkowski et al. successfully created the first 4n:2n mosaic mouse by exposing 2- to 4-celled embryos to cytochalasin B. Surprisingly,
less than 4% tetraploid cells were observed in embryo proper whereas approximately 50% tetraploid cells werepresent in the extraembryonic tissues (Tarkowski et al., 1977). Another study showed that only 3% tetraploidcell contribution in the bone marrow of a 4n:2n chimera (Lu and Markert, 1980).
To assess the contribution level of the embryonic tetraploid and diploid cells in a chimera, ICR (Institute ofCancer Research) female Mus musculus were crossed with two different male strains: red fluorescent H2BmCherry (Abe et al., 2011) and green fluorescent H2B-GFP (Kurotaki et al., 2007). GFP, a green fluorescentprotein, was first discovered by Shimomura et al. in 1962; it has become an important tool to track and visualize
the proteins of interest because of its ability to binding to the C-terminus of histone H2B (Kanda et al., 1998).
mCherry is a GFP derivative that emits red fluorescent.
This experiment aimed to investigate the contribution level of tetraploid cells in a chimeric embryo. The diploid blastomeres were electrofused to form tetraploids, and then they were aggregated with the diploid embryonic cells to form chimeras. After 24 hours, one 4n:2n aggregate chimera was produced, and it was used fortime–lapse analysis.

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MATERIALS AND METHODSThe experiments were performed in accordancewith the following procedures described in the experimental manual with several modifications (Nagyet al., 2003).
Flushing and Collecting Embryos
Before flushing the embryos, prepare a culturing
medium using KSOM. Place several KSOM drops ona small dish and cover them with mineral oil. Placethe medium in 37°C and 5% CO2 incubator beforehand.
After isolating a pair of fallopian tubes from amouse, M2 medium (preferably pre-warmed to 37°C)
was used to temporally soak the tubes and embryos during the process. Terumo’s® 1 mL Tuberculin
syringe containing M2 was used with a number 30needle.
A stereomicroscope (Leica® MZ-16) was used tolocate the infundibulum of uterine tube that is situated at the end of the fallopian tube. The infundibulum was held using No. 5 tweezers, the needle wasinserted into it, and then embryos were flushed out
from the uterus. These embryos were collected withcapillary pipettes and then washed twice in M2 medium. Finally, they were gently placed in the KSOM
media plate and incubated in 37°C.
Electrofusion of the Blastomeres
Sufficient amount of mannitol (0.3 M) was placedin the slit of the electrode chamber, and the chamber FIGURE 2. PROGRESS OF ELECTROFUSION
The image on the top shows the 2-celled embryos before the electrofuwas connected with the pulse generator (SonielTM sion and the one on the bottom shows the fused embryos 20 minutes
Electroporator CUY21 EDIT). Approximately 10 em- after the electrofusion fusion.
SOURCE: EPPENDORF
bryos (at once) were placed in the slit slowly andtheir blastomeres were carefully positioned perpendicularly to the electrodes. After operating the electricalpulse (100V), the embryos were collected and washed with KSOM before being incubated. The blastomere
fusion can be observed whthin one hour (Figure 2).
Formation of Aggregation Chimeras
Multiple KSOM drops were placed on a 60-mm plastic dish plate. At the centre of each drop, a small
depression was created with a 1-mm round-tip pin (preferably a Japanese “machi-bari”). Finally, all drops
were covered with liquid paraffin. Several embryos were placed in acid tyrode to dissolve their zona pellucidae and immediately placed in M2 solution (with HEPES buffer) to neutralize acid. The embryos werethen washed several times in KSOM solution. Each tetraploid embryo and diploid embryo was assigned tothe same depression to form an aggregation. Finally, the aggregated chimeras were incubated (37°C/5%CO2) for 24 hours.
Time-lapse Imaging of the Development of Chimeras
Yokogawa’s time-lapse image capturing device Cell VoyagerTM was used for capturing images of 500 time
points at 10-minute intervals. The images obtained from this process were then modified into movie clips
and colored images to visualize and analyze tetraploids (GFP) and diploids (mCherry).

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RESULTS
Dissecting Mus musculus and flushing out embryos from fallopian tubes:
On July 13 (group 1), the first two female mice weredissected approximately 1.5 days post coitum (dpc),
and another two female mice were dissected after fertilization. Twenty-eight embryos were collected from thefirst two mice; three embryos were collected from thelatter two.
On July 15 (group 2), 23 embryos were collectedfrom two H2-mCherry (mCherry)-crossed mice and 27
embryos were collected from H2B-GFP (GFP)-crossed
mice around noon. Approximately four hours later, 26
embryos were collected from the other two mCherry and25 were collected from two GFP mice (Table 1).
On July 21 (group 3), embryos were collected fromseven mice (three mCherry and four GFP). From the84 embryos that were collected, 42 embryos each werecollected from mCherry and GFP mice.
On July 27 (group 4), 33 embryos were collected fromtwo mCherry mice and 52 embryos were collected fromthree GFP mice. Of the 33 mCherry embryos,
26 were	  2-celled, six were 1-celled, and one embryo was abnormal, whereas all 52 GFP embryos were 2-celled.

Table 1. Amount and condition of embryos at about 1.5 dpc.

Most embryos demonstrated their 2-cell stages. 1-cell embryos possibly indicate the failureof fertilization. Only one 3-cell embryo was found between 1.5 to 2.0 dpc.

Condition of Embryos
1-Cell2-Cell3-CellJuly 13, 2011 (Out of 4 Mice)
N/A
30
1WT
July 15, 2011 (Out of 8 Mice)742
N/A
mCherry
GFP
N/A52
N/A
July 21, 2011 (Out of 7 Mice)1032
N/A
mCherry
1131
N/A
GFP
July 27, 2011
mCherry
626
1
N/A
52
N/A
GFP
August 3, 2011
mCherry
1433
N/A
GFP
1031
N/A
August 10, 2011 (Out of 7 Mice)mCherry
729
216
30
N/A
GFP

MiceTotal
3149
52
4242
33
52
47
4138
46	  On August 3 (group 5), 47 mCherry embryos were collected of which 33 were 2-celled and 14 were 1-celled; 41GFP embryos were collected of which 10 were 1-celled and 31 were 2-celled.
On August 10 (group 6), 38 mCherry embryos and 46 GFP embryos were collected. The significances of eachstrain are also described in Table 1.
Development of Diploid (2n) Embryos:
All embryos were cultured in vitro: KSOM was used as the medium and a water-jacket incubator was set to maintain constant temperature (37°C) and CO2 level (5%).
Group 1 (July 13) embryos were again observed under a microscope at approximately 2.5–3.0 dpc. In total, therewere thirty-one embryos: 12 embryos in their compaction stage, one embryo in 4-celled stage, and the remaining
in multi-celled (5-to 8-celled) stage.
In group 2 (July 15), only few embryos were observed at 2.5 dpc because 10 mCherry and twenty GFP embryos
were used for time-lapse analyses. At 2.5 dpc, among 25 diploid (2n) mCherry embryos, four embryos exhibitedcompaction, 20 were in multi-celled stage, and one embryo was found dead. Meanwhile, all 16 2n GFP embryos
were in multi-celled stage. Since the 2n GFP embryos were controls, they were not carefully observed throughout
the experiment; however, they were examined at 4.5 dpc, and it was confirmed that the embryos had progressed
towards the blastocyst stage. Because of inefficient chimera aggregation, no 2n mCherry was remained.
The group 5 (August 3) embryos did not fully grow due to high concentration of the growth medium (KSOM);
however, the embryos in group 6 (August 10) grew uneventfully and 30 healthy GFP embryos were ready to beelectrofused.
Health Conditions of Electrofused Embryos:
Fourteen 2-cell mCherry embryos and 15 2-cell GFP embryos were selected to undergo electrofusion to produce
tetraploids. At 2.5 dpc, one 2-cell, four 3-cell, six 4-cell, and three multi-celled mCherry tetraploid embryos wereobserved; five 2-cell, four 3-cell, three 4-cell, and three multi-celled embryos were observed in GFP tetraploids.

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In group 2, 30 mCherry embryos were electrofused and incubated in vitro. Approximately 3 hours later, the blastomere of only one embryo was verified to be fused because it was completely 1-cell whereas other artificially-fused
embryos were 2-cell stage.
Out of 30 GFP embryos, only 16 of them were found fully fused after one hour from electrofusion. As for the unfused embryos, the electrofusion procedure was repeated and then they were cultured overnight.
Significance of Aggregated Chimera:
We attempted to produce nine aggregation chimeras in group 3; however, only one set of embryos was seemed
to be successfully aggregated. Fluorescent microscopy showed that the aggregated embryos emitted high intensity
green light and low intensity red light. Therefore, one chimera had surely been produced. Despite the successful aggregation of one set, all the embryos (including the chimeric one) did not seem to grow. The embryos in thecontrol group (2n GFP embryos) had already progressed to the blastocyst stage, whereas the embryos of the aggregated chimeras were still at the 4- to 8-cell stage.
In group 6, 14 healthy tetraploid GFP embryos (out of 16) were used to produce aggregation chimeras with diploid mCherry. 13 successfully aggregated 4n:2n chimeras were obtained consequently.
Visualizing a 4n:2n Chimera (Group 6 Exclusive):
Twelve aggregated 4n:2n chimeras were selected
to be visualized by time-lapse. The time-lapse image
showed the distribution of tetraploids (green) and diploids (red) as shown in Figure 3. This image was taken
at t = 210 (1.46 days after the first time-lapse image wastaken), and it appeared that although the tetraploids anddiploids had aggregated, they had not completely mixed
up. The size of the nuclei of tetraploids seemed to bealmost double that of the diploids, which would be reasonable considering that the tetraploids contained twice
as much as chromosome pairs than the diploids.

BRIEF DISCUSSION
Analysis of time-lapse images of six of twelve chimeras
has revealed several noteworthy phenomena.
In this experiment, the visualization and analysis of thetime-lapse images were slightly challenging because ofuncertainties regarding the inner cell mass and the trophectoderm regions. Therefore, dyeing the trophectoderm
FIGURE 3. DIPLOIDS AND TETRAPLOIDS IN AN EMBRYOcells (possibly with Cdx2) to differentiate them from the The green cells (GEP) represent tetraploids whereas the red cells
ICM cells would prove effective in further studies.
(mCherry) represents diploids. The image was captured at 2100 minThe distribution and contributions of the tetraploids and utes after the time-lapse image was taken
diploids in embryos have a particular pattern. The movement of tetraploids in the embryo is intriguing and might provide explain the studies conducted by Tarkowski et al. (1977) and Lu and Markert (1980).

ACKNOWLEDGMENT
I would like to thank Professor Fujimori and the people at the Fujimori Lab for their support both materially and academically. I would also like to express my gratitude to the National Institute for Basic Biology for this memorable andintellectually stimulating internship experience.

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