Connor Wang

BIOL 4070-2
Fall 2013
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Title
Role of convergent extension in increasing or decreasing Xenopus laevis blastopore
closure rate under varying temperatures and salt concentrations
Abstract
During embryonic gastrulation, vertebrate embryos undergo dramatic changes in shape
and morphology. Convergent extension is one of the motors driving blastopore closure (Keller et.
al., 2000). Development of Xenopus laevis embryos under different temperature treatments has
been well documented; it is known that embryos tend to develop slower and reach gastrulation at
later time periods and at low temperatures than compared to treatments at higher temperatures
(Khokha et. al, 2002). Here, we seek to elucidate the role of convergent extension in driving
blastopore closure in Xenopus laevis. We measured the area of the yolk plug as well as delay in
blastopore closure in embryos treated under various combinations of temperature and salinity
concentration. Additionally, we allowed embryos to develop into tadpoles in order to measure
their anterior-posterior axis lengths, which revealed the role of convergent extension in driving
blastopore closure. We maintain that by changing the temperatures and salt concentrations, we
can effectively alter the rate of blastopore closure due to convergent extension in Xenopus laevis
embryos.
Introduction
The model system used in our experiments is the early embryos of Xenopus laevis. Here,
we studied the role of convergent extension in driving blastopore closure during Xenopus laevis
gastrulation. We did this by observing the effects of changes in temperatures in association with
changing salt concentrations on the speed and involution of blastopore closure, by measuring the
area of the yolk plug, and by recording the anterior-posterior axis lengths of Xenopus embryos at
tadpole stages, as staged by the Nieuwkoop and Faber staging series (Nieuwkoop, 2005).
Anterior-posterior axis lengths serve as a measure for the role of convergent extension, a
mechanism by which the dorsal structures of the embryonic vertebrates elongate. Should
convergent extension be made slower or have defects in elongation during embryonic
gastrulation, the organism will present a corresponding anterior-posterior axis that is shorter than
expected (Wallingford et. al., 2002). Thus, if convergent extension is affected here, it may be due
to treatments under different temperatures and salinity concentrations.
Here, we placed particular focus on the process of gastrulation, the phase of development
where the marginal zone is internalized, simultaneously closing the blastopore and elongating the
anterior-posterior axis. The movement of gastrulation establishes the three layered structure with
the ectoderm, mesoderm, and endoderm; it also involves numerous morphogenetic changes in
the embryos such as cell migration and the force-producing cell intercalation (Keller et. al.,
2008). The onset of gastrulation is marked by the appearance of apically constricting bottle cells
on the dorsal side, opposite of the sperm entry point (Slack, 2001). This represents the dorsal lip.
The blastopore then becomes a circle where the dorsal lip is the dorsal segment of the complete

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circle and the ventral lip is the ventral part. Deep marginal cells of the embryos invaginate all
around the blastopore (Slack 2001).
In the course of invagination of the involuting marginal zone during gastrulation, the
archenteron becomes the principal cavity and the embryo rotates in such a fashion to position the
dorsal side up, presenting a true anterior-posterior axis that runs from the leading edge of the
mesoderm at the anterior to the posterior (Keller et. al., 2008). In our experiment, a common
theme frequently encountered under treatments of high salt concentration is the evagination of
the ectoderm known as exogastrulation. Instead of involution, the germ layers evaginate to form
a ball-like structure outside the embryo. The germ layers remains normally patterned while the
central nervous system is defective (Slack, 2001). We induced exogastrulation in embryos treated
with high salt concentrations; however, we found that this phenotype may be rescued by
temperature regulations.
It is important to note that convergent extension is only one of the components of the axis
elongation machinery. For example, directed migration of the head mesoderm also contributes to
the elongation of dorsal mesoderm in Xenopus. Additionally, spreading of the animal
hemisphere, called epiboly, leads to it eventually covering the entire embryo. Although these
morphogenetic processes are distinct from convergence extension, they no doubt play significant
roles in axis elongation (Wallingford et. al., 2002).
During vertebrate gastrulation, there are coordinated cell movements that shape the basic
body plan of the future organism, as described above. Within such development is the role of
convergent extension in gastrulation; convergence and extension is a highly conserved
morphogenetic movement that takes place in the mesoderm. It is a universal method of
coordinated movements of individual cells in vertebrate morphogenesis. It involves the cells of
many embryonic embryos to narrow in one dimension while lengthening perpendicularly in
another dimension by mass cellular movement (Wallingford et. al., 2002). During gastrulation,
the main forces driving convergent extension are due to the tissues that exist within the
involution marginal zone of the embryo. This zone lies between the vegetal endoderm and the
posterior neural tissue. The involuting marginal zone rolls over the blastopore lip and turns
inside-out. As it turns, the zone converges and extends to narrow the germ layers mediolaterally
and elongate the embryo from the posterior to the anterior (Keller et. al., 2008). The rates of
convergence and extension are shown to differ greatly according to the position and stage of
gastrulation. Here, we observed whether changes in temperature as well as salinity
concentrations underlie changes in the rate of blastopore closure due to convergence and
extension.
Several investigators have previous conducted studies on the deviation in body
proportions of Xenopus embryos under varying temperature or salt concentrations; however, our
research provides an in depth look at both temperature and salt concentrations together as factors
in rearing Xenopus laevis (Hilken et. al., 1995). Our preliminary experiments provided intriguing
results that have acted in accordance with our hypothesis. Our independent variables for our
experiments were temperature treatments and salinity concentrations.

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Materials and Methods
Our preliminary experiments involved generating sets of pictures from microscopes
based on differing temperature and MBS concentrations. We first obtained embryos that were at
stage 6. The staging of the embryos is done using the Nieuwkoop and Faber staging series
(Nieuwkoop, 2005). Stage 6 embryos were chosen as the base point because they are at the stage
at which the embryos are most easily de-jellied. We de-jellied the embryos by immersing them in
cysteine solution accompanied by xMR rinsing. The SH side groups on this amino acid solution
will break down the disulfide bonds of the jelly proteins, which can then be washed off. The eggs
were swirled about in the dish until they lie close to one another, which indicates the jelly coat
has been removed.
We conducted experiments on 3 different temperature points (12 degrees Celsius, which
is generally considered very cold for Xenopus, 18 degrees Celsius, and 25 degrees Celsius) and 6
different MBS concentrations (0.1X, 0.25X, 0.33X, 0.5X, 0.8X, and 1.0X MBS). The control
dish for each temperature set was the dish with embryos in the 0.33X MBS. The temperatures of
the tanks were stable within 1 degree Celsius. Each temperature set is placed into their respective
incubators at the same time, and the incubation time to reach stage 11 is measured using
developmental rate equations (Eq 1 & 2) extrapolated using linear regression from previous data
where x is the hours since fertilization and y is the stage of development. Each temperature set is
promptly fixed in MEMFA when the control group of 0.33X MBS reaches stage 11, regardless
what stage the other saline concentration treatment groups are at. We then measured the
blastopore opening of each picture using Image J, keeping consistent the number of embryos
captured per image.
Eq 1. Developmental Rate at 18 degrees Celsius: y=0.3474x + 4.8374
Eq 2. Developmental Rate at 12 degrees Celsius: y=0.1877x + 4.0575
In addition to stopping at stage 11 in embryo development, we also stopped several
treated embryos at stage 28, identified using by prominent fin divided between outer and inner
bands (Nieuwkoop, 2005). The embryos here were measured for their anterior-posterior axis
lengths.
Exogastrulation is a common phenotype observed under high salt treatments (Slack,
2001). Exogastrulated embryos were taken into account and counted when viewed under the
microscope.
In setting up for a time-lapse microscopy, we made a unique dish to place the embryos in
and followed them through gastrulation. The dish was constructed with a small petri dish divided
into three sections with coverslips and oil. Stage 6 embryos were placed into small openings
present on a sheet that was put into the petri dish. Respective MBS concentration was allocated
to each section. MBS concentrations tested were: 0.1X, 0.33X, and 0.5X; 0.1X and 0.33X; 0.33X
and 0.8X MBS. The video was retrieved and key moments in gastrulation were documented and
measured using Image J. During filming, the embryos often flipped over to present the dorsal
side up. When this occurred, we intervened and flipped the embryos back to the desired position.

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Additionally, instead of measuring yolk plug area, we measured blastopore diameter, as some of
the yolk plugs were difficult to measure.
Furthermore, immunocytochemical methods were employed to stain for notochord and
mesoderm. First vitelline envelopes of stage 6 embryos were removed with watchmaker forceps
and fixed in MEMFA overnight at 4 degrees Celsius. The embryos were then rinsed with PBS
and blocked in PBT-GS for 2 hours at room temperature. Afterwards, primarily antibodies were
added, and embyos were incubated overnight at 4 degrees Celsius. We then washed them in PBT
and incubated them with secondary antibody appropriate to our primary antibody. The vials were
kept from light by shielding with a piece of tin foil. The resulting embryos were washed with
PBT, re-fixed in MEMFA, and washed with 100% methanol. To view under the fluorescence, the
methanol was replaced with Murray clear.
Results
TheEffectofSaltConcentrationonBlastoporeClosureat3Temperatures

Normalizedareaofyolkplug

3
2.5
2
1.5
1
0.5
0
0.1 0.25 0.33 0.5

12 degrees C
18 degrees C
25 degrees C
0.8

MBSconcentration

Figure 1. The effect of salt concentrations on blastopore closure at three temperature points.
Our final data generated in Figure 1 represents the effect of salt concentrations, as
represented by MBS, on blastopore closure at three temperature points. The results indicate that
at 0.8X and 1.0MBS for all temperature points, blastopore closure was delayed. Blastopore
closure delay was measured by yolk plug area, as the larger yolk plug area indicates that
gastrulation has not progressed as far than those embryos exhibiting smaller yolk plugs. 5 trials
of 18 plates each were done for each experiment.
In addition to measuring pictures taken on the computer after fixation, we sought for
more accurate measurements of blastopore closure by use of time-lapse microscopy. This is done
because, though we observed a certain morphological phenotype in the embryos after fixation,
we were not certain that there are no other factors that may be influencing the eventual
phenotype that we are measuring.

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APAxisLengths
1.5
1
0.5
Pixelnormalizedtopixelof0.33XMBS
0

25 degrees

MBSconcentration

Figure 2. Figure 2 delineates the anterior-posterior axis lengths measured for the differing MBS
concentration treatments at various temperature points.
According to figure 2, only pictures for the 18 degree embryos proved to be helpful at
stage 28; pictures taken of the embryos at other temperature points were inconclusive because
the embryos were of odd, rotund shape and exhibited irregular anterior-posterior axis, as shown
in Figure 3 and Figure 4.

Figure 3&4. Stage 28 Xenopus laevis embryo at 12 degrees Celsius and 0.8X MBS and at 25
degrees Celsius and 1.0X MBS, respectively.

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Figure 5. Figure 5 exhibits the exogastrulation percentage at the three temperature points.
Exogastrulation appearance only occurred at 0.8X and 1.0X MBS, which was consistent
with current understanding of exogastrulation. There were drastically lower percentages of
embryos that exogastrulated at 18 degrees when compared to extreme temperatures.
Discussion
We postulated that decreased concentration of MBS but still above 0.25X and increased
temperature will have a stimulatory effect on the development of the embryos, but these
conditions may lead to malformations of certain body parts in the final organism due to drastic
effects on the proteins during development. An example of such malformation is exogastrulation.
Additionally, Nelson et. al suggests Xenopus laevis embryos may grow slower at decreased
temperature conditions due to correlations with membrane fluidity. They maintain that
poikilothermic animals have been observed to adapt themselves to changing temperatures by
modifying the proportion of unsaturated fatty acids in the cell membranes (Nelson et. al., 1982).
However, in another research study conducted by Hilken et. al, it was discovered that
ectothermic animals, such as the embryos of the frogs studied here, showed no differences in
growth development at varying water temperatures over a five month period. They did observe
that frogs kept in water of 19 degrees Celsius grew slower than the others kept in higher
temperatures for the first fourth months, but subsequently surpassed those kept in higher
temperatures in body weight and length in the last month of observation (Hilken et. al., 1995). As
such, we hope to include more dependent variables in our research such as development of the
body length and weight of the embryos and also carry the development of the Xenopus laevis
embryos into the tadpole stages in order to study the thermosensitivity of tadpoles or their
swimming abilities under optimal salt concentrations (Nelson and Sillar, 2009).
To interpret the results from our measurements, we normalized each data value to 0.33X
MBS data values, which represents the control for each temperature set of the experiment. To do
this, we divided each data value by the average area of the yolk plug obtained for 0.33X MBS of
each temperature set. This normalization also allows us to compare different spawning of
fertilized embryos, since different spawning may result in embryos of larger sizes due to
differences in the amount of vegetal endoderm. The lower the ratios obtained, the further along
in development of the embryos, because it meant that the blastopores have closed the most and
are the most ready to move onto the next stage in development.
Figure 1 also exhibits that the best MBS for blastopore closure when temperatures are at
the extremes (25 degree Celsius and 12 degree Celsius) is at 0.5X MBS. For 18 degree Celsius,
0.33X MBS groups exhibited the lowest blastopore closure delay. This data suggest that when
there is stress due to temperature, a higher salinity may be able to compensate in terms of
convergence and extension and help the embryo close its blastopore. The data obtained from
these experiments are robust because five trials were completed. Each experiment consisted of 3
plates representing each temperature for each of the 6 different MBS concentrations; however, a
disadvantage of some of our trials is due to inconsistency in the sample size for each plate
causing an increase in variation. This occurred because of the fickle nature of the embryos,

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causing many to die within a certain dish while many to live in another dish; this interferes with
our efforts to keep the sample size consistent across measurements.
To ascertain the path the embryo is taking early in development and during the treatment
conditions, we employed time-lapse microscopy to follow the embryo as it goes through
gastrulation. For example, if the embryos, under changing environmental conditions, are acting
to try to close their blastopore, thus generating counteracting forces in the process, it could create
interesting research avenues for future projects. We also sought to perform
immunocytochemistry to stain for appearance of notochord in stage 28 tadpoles. We hoped to do
this because if our treatments affected convergence and extension, then we should observe a
wide notochord in the resulting tadpole, in addition to a shorter anterior-posterior axis
(Wallingford, et. al., 2002). However, the data procured from the time-lapse video provided no
reliable or sensible results. This occurred because the embryos, though de-vitellined, did not stay
stagnant and flipped over constantly. To fix these problems in the future, we suggest that there be
a way for the embryos to remain motionless as they develop or to check the embryos periodically
in order to ensure that the correct side is facing the camera. Additionally, we recommend having
more embryos per frame to increase robustness of the trial.
Results from figures 2-4 suggest that high saline concentration may be detrimental to the
function of convergent extension. Anterior-posterior axis formation failed in both 12 degree and
25 degree Celsius treatment under high salinity concentrations because the embryos did not
proper extend. This suggests that convergence and extension did not play a major role in
embryonic development here. A downside to the results procured from this experiment can be
attributed to the lack of multiple trials
Moreover, there was drastically lower percentages of embryos that exogastrulated at 18
degrees when compared to extreme temperatures, suggesting that temperatures may play a role in
exogastrulation, in addition to current understanding that high salt causes exogastrulation. Our
results also suggest that we may be able to compensate for high salinity concentration by
developing the embryos at moderate temperature, thus recuing embryos from exogastrulating.
This result is extrapolated from multiple trials.
Modifications we can make to better refine our results are: make sure to keep the
population density of the embryos on each plate consistent while also taking into account
possible embryo death; doing greater number of trials; having more replicates of each plate;
doing more temperature treatments; and better timing in terms of staging and fixing the embryos.
As stated above, we also hope to include more dependent variables into our results - ones that
will help us gain a greater understanding of how temperature and salt concentrations effect
embryo development and growth.
Additionally, there have been recent experiments reporting that a non-canonical Wnt
signaling cascade also participates in the regulation of convergent extension movements in
Xenopus (Ohkawara et. al., 2003). This is an interesting avenue of research that could be
combined with our experiments here. It would be interesting to see if knocking down the
expression of Dishevelled (Dsh), which acts downstream of the Wnt cascade will have any effect
in modulating blastopore closure (Djiane et. al., 2000). Another thought we had hoped to explore

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are to try conducting this experiment instead with Xenopus tropicalis as the model system
instead. Moreover, as mentioned above, there are a great many morphogenetic processes that
come together during gastrulation to modulate development of the Xenopus embryo (Slack,
2001), as a result, for future research, it may be worthwhile to continue to look at all the possible
factors and cellular mechanisms which contribute to formation of the vertebrate embryo.
Acknowledgements
I would like to thank Jennifer ONeal, Sammi Kim, Daniel Song, and Osei Appiagyet of
team Mo Bedda for their time and their incredible company this semester. I would like to give
special thanks to Katherine Pfister who worked and helped us tirelessly with preparing the
embryos, coming up with ideas for this project, fielding the teams wild questions patiently,
reading my proposal, and proof-reading this final paper. I would also like to acknowledge Dr.
Raymond Keller for his extensive help in this project, for reading my proposal, for providing
valuable feedback, and for being available when the team encountered problems. I would like to
thank Kay for helping us prepare the reagents that made this project possible.
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