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Abstract:
Alx1 is a beta catenin dependent homeodomain protein; its expression is necessary for
skeletogenesis and ingression of primary mesenchyme cells (PMCs) in Lytechinus variegates
(Lv). Previously, Alx1’s role in these processes was determined by blocking its expression using
morpholino antisense oligonucleotides, which prevented both ingression of PMCs and
skeletogenesis. However, this approach can only be used to determine Alx1’s function during
early development because these oligonucleotides are injected at fertilization resulting in
persistent repression during development. Our goal is to obtain a greater understanding of Alx1’s
function throughout development by temporally controlling its expression. We generated a DNA
construct encoding LvAlx1 fused to green fluorescent protein (GFP), and the glucocorticoid
receptor (GR) ligand binding domain. Addition of the GR ligand binding domain allows Alx1
localization to be regulated. In the absence of the hormone ligand dexamethasone, the ligand
binding domain traps the fusion protein in the cytoplasm by causing it to bind to a cytoplasmic
heat shock protein (HSP). Upon addition of dexamethasone, the fusion protein dissociates from
the HSP and entered the nucleus where Alx1 is able to activate the transcription of other
skeletogenic genes. The green fluorescent protein allows any subcellular migration of Alx1 to be
observed. Dexamethasone was added to eggs that had already been injected with the mRNA for
the fusion protein at various embryological stages. It was found that this system is both feasible
and efficient in sea urchins for controlling the location of the fusion protein in the cell, and
promises to be useful for manipulating Alx1 activity.
Introduction:
Sea Urchins as a Model Organism: There are several reasons for studying
skeletogenesis in sea urchins as opposed to other genetic model organisms. First of all, sea
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urchin eggs and embryos are relatively easy to work with for a complex animal. This is because
they are transparent, allowing all stages of development to be visible. During the early stages,
the nucleus is also visible. Also, they produce a relatively small number of cells, which makes it
easier to keep track of cell lineage and developmental stages. Sea urchin embryos are easily
available and they develop rapidly in sea water. Eggs can be easily obtained from adult sea
urchins in vast quantities. Once the eggs are fertilized, depending on the species and water
temperature, the zygote will reach the larval stage within a day or two. Another reason the sea
urchin is a good model for skeletogenesis is that when exogenous DNA is injected into the egg
cytoplasm, it is rapidly taken up and incorporated into the embryonic genome. This incorporated
DNA allows genes of interest to be expressed during development, further facilitating the use of
sea urchins as a genetic system.
Sea urchins are also ideal for genetic studies because despite their simplicity, they are
close relatives of humans. In fact, they are a closer relative to humans than any other nonchordate animal, including those commonly used for study such as the fruit fly or nematode,
because they are a member of the Echinoderm phylum which makes them deuterosomes along
with humans1. Flies and nematodes used to have the advantage over sea urchins because their
genomes were sequenced first. However, the sequencing of the genome of the sea urchin
species, Strongylocentotus purpuratus was completed recently, making sea urchins even more
ideal for genetic studies1.
Overview of Sea Urchin Development: The previously mentioned features allow the
Primary Mesenchyme cell (PMC) lineage of the sea urchin embryo to be a model system for the
analysis of fate specification, cell signaling, morphogenesis, and biomineralization. A brief
overview of how a sea urchin embryo develops is helpful in explaining these processes2. First,
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the egg is fertilized, as shown in the first frame of Figure 1. Note that the nucleus appears as an
indented region near the center of the fertilized egg. Due to an uneven distribution of certain
proteins and maternal mRNAs in the egg, the egg begins to divide unevenly, creating an animal
and vegetal tier that will give rise to different types of cells. The vegetal tier, which makes up
the bottom half of the embryo in the 16-cell stage shown in Figure 1, divides unevenly to
produce macromeres and micromeres. The macomeres are the larger cells, while the micromeres
are the very small cells at the very bottom of the 16-cell embryo. The micromeres will divide
unevenly again, creating large and small micromeres.
Primary Mesenchyme Cells: The large micromeres produce the primary mesenchyme cells
(PMCs). The PMCs ingress and eventually become the skeleton of the embryo2. Note that at the
mid-gastrula stage, shown in Figure 1, there are some small clumps of cells that have gone into
the hollow part of the embryo. These are PMCs. By the late gastrula stage they have formed a
ring around the coelem of the embryo. They will secrete calcium carbonate at this point to make
spicules, the skeletal elements found in the pluteus larvae. Another function of PMCs is signaling
of vegetal invagination (seen as the bottom cells folding inwards at the early gastrula stage,
frame 4 of Figure 1). Interestingly, if the PMCs are removed, another cell type will adopt the
PMC fate and become the skeleton. That is, non-PMC cells will take over the role of the missing
PMCs.
These processes are autonomous in that PMCs will still form skeletal elements on their
normal time scale even if removed from the embryo and grown in isolation. It has been found
that this behavior requires a functional Raf/MEK/ERK signaling pathway that is activated in a
cell-autonomous manner3. This path results in the phosphorylation of transcription factors by
ERK, which allows genes required for differentiation to be transcribed without signaling from
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other cells. Therefore, PMCs do not require signaling from other cell types to perform their
function.
Figure 1: Stages of Sea Urchin Development. PMCs are indicated by arrows where
relevant. A. Fertilized Egg. B. 16-cell stage. C. Mesenchyme blastula. D. Early gastrula. E.
Mid Gastrula. F. Late Gastrula. G. Late gastrula. H. Pluteus larva
Skeleton formation in sea urchins occurs once the PMCs have ingressed and formed a
ring. The skeleton of the pluteus larvae is made up of long filaments called spicules. The PMCs
deposit this skeleton, which is composed of a branched network of magnesium calcite rods and
spicule matrix proteins. A sulfated cell-surface glycoprotein that has been implicated in calcium
uptake or deposition, as well as several other PMC-specific gene products that participate in the
synthesis of the skeleton have been identified4. The entire process, including the formation of
PMCs in the early stages of development, is controlled by a gene regulatory network. β-catenin
a maternal transcription factor, referred to as such because it is present in the unfertilized egg, is
essentially the “on” switch for the network, as shown in Figure 2. The Beta catenin activates the
gene for the protein Pmar1, which is a transcriptional repressor of global repressor HesC. Pmar1
is necessary for initiating the micromere specification program and for the expression of
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inductive signals produced by micromeres5. In the absence of Pmar1, HesC prevents the
transcription of other genes involved in skeletogenesis, but once Pmar1 is active, the rest of the
network is free to operate and create a skeleton6. Every cell in the sea urchin has the same genes,
but not every cell becomes skeleton-forming. This is because the network allows for the
repression or expression of skeleton forming genes depending on the presence of β-catenin. βcatenin is only concentrated in a certain area of the unfertilized egg, and then is segregated into
only the cells that this area gives rise to.
Figure 2: The Micromere-PMC gene regulatory network 6,7
Alx 1’s Role in the Gene Regulatory Network: An important member of the PMC gene
regulatory network is Alx 1, which is a homeodomain protein and transcription factor. This
protein is expressed exclusively by large micromeres, implicating it in skeletogenesis and
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legitimizing study of the gene as a member of the gene regulatory network7. Previous findings
verify that Alx1 is required for skeletogenesis and is a member of the network. Alx1 controls
genes required for epithelial-mesenchymal transition (PMC ingression) and biomineralization
(skeleton formation). Using in situ hybridization, it was found that levels of Alx1 were high in
PMCs following ingression and then gradually declined until late gastrula stage where the
mRNA for this protein was no longer detectable7. It was determined by morpholino
oligonucleotide studies and over-expression studies that Alx1 is essential at an early stage of
specification7. Morpholino oligonucleotides (MOs) bind to complementary mRNA, preventing
the mRNA from becoming transcribed into protein. Blocking Alx1 expression, through the use
of MOs causes delayed invagination, no ingression of PMCs, and failure to form skeletal
elements7. Over-expression is induced by flooding the cell with excess mRNA for the protein of
interest. Recent data from Dr. Charles Ettensohn’s lab has shown that over-expression of Alx1
causes an increase in the number of PMCs, but the exact point during development at which
Alx1 exerts this effect is unknown.
In order to study Alx1 at the point in which it naturally acts, further study is required, as well
as a new technique. The problem with injection of mRNAs and morpholinos is that they cause
immediate and widespread changes in expression. While these techniques were convenient for
determining the overall functions of Alx1, it is important to be able to study the protein when it
expressed at the appropriate time. The previous methods cause skeleton to be formed in excess,
or not at all, which may have dramatic effects on other aspects of the embryo’s development.
They also cause effects so profound that it is difficult to determine which more subtle roles Alx1
might play in development. It would also be beneficial to study expression of Alx1 at a later
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than natural time, which could be helpful in determining unknown roles Alx1 has in
development.
A Hormone-Inducible System for Temporally Regulated Study of Alx1: A HormoneInducible System is one technique that confers the ability to induce gene expression in a
temporally controlled manner. This technique has been used successfully in a whole organism to
study Xenopus laevis (frog) development. The Xenopus system used a fusion protein between the
hormone-binding domain of the glucocorticoid receptor (GR) and their protein of study, MyoD,
a transcription factor needed to express muscle-specific actin8. Our L. variegatus system used a
fusion protein between the GR ligand binding domain, Alx1, and green fluorescent protein,
which allows for visualization of the migration of the fusion protein. In the absence of the
hormone, dexamethasone, the fusion protein is sequestered by heat shock proteins in the
cytoplasm, keeping the transcription factor, Alx1, inactive.
After hormone is added, the fusion protein will theoretically be able to enter the nucleus.
At this point, the protein of interest, in this case a transcription factor, is able to exert its function.
Because a transcription factor must be able to access the nuclear DNA to function, it is inert
while trapped in the cytoplasm. The hormone was found to act quickly enough for the system to
be useful in the study of frog transcription factors8. Because our protein of interest is also a
transcription factor, and our model organism is also aquatic, which would allow the hormone to
be applied in the same way, this technique promised to be feasible for studying Alx1. Our goal
was to determine if the system also works in sea urchins so that it can be implemented to study
Alx1 in a temporally controlled manner.
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Materials and Methods:
Cloning: The first step in implementing the hormone-inducible system in sea urchins was
the creation of a DNA construct. This was needed to make mRNA in vitro, which would then be
used to produce the fusion protein in vivo. We used PCR to generate Alx 1 and GR ligand
binding domain fragments from existing pCS2+ vector constructs. This vector includes a variety
of promoters and restriction enzyme sites. The pieces were digested with Cla I and BamH I
restriction enzymes and ligated to obtain the order: Alx 1, glucocorticoid receptor, GFP. This
construct will be abbreviated as Alx1.GR.GFP. Similar constructs made previously in the
Ettensohn lab showed that this particular order was needed for the most efficient fusion protein.
Colony Forming Unit PCR was used to screen for Escherichia coli colonies that were
transformed with the Alx1.GR.GFP construct, and that had the insert in the correct orientation.
The pCS2 vector conferred resistance to ampicillin. Therefore, E. coli cells were transformed
with the ligation product and grown overnight at 25˚C on a plate of media containing ampicillin.
Then 25 colonies were chosen at random for screening. Most of each colony was deposited in a
small PCR tube containing the necessary buffers and enzymes as well as primers complementary
to the pCS2 vector and the ALX1 insert. The colonies were also streaked onto a master plate and
were allowed to continue growing. After completing PCR, gel loading dye was added to each
sample and they were analyzed using gel electrophoresis. Only colonies transformed with pCS2
vector plus the Alx1 and ligand binding domain fragments would produce DNA fragments
during PCR in large enough quantities to be seen on the gel. The bands were also checked for
appropriate size: it was determined that the band produced by the vector with the insert in the
correct orientation would be 1800 base pairs long. In Figure 3, three such colonies were found
and can be seen in lanes 5, 6 and 17.
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Figure 3: Image of Colony Forming Unit PCR gel. The bands labeled at 1800bp are PCR
products resulting from colonies transformed with the complete Alx1.GR.GFP construct. The
blurred bands at the bottom of each lane are primer dimmers.
The construct was produced and collected in mass from each of three colonies shown in
Figure 3 by growing a large culture of each colony and then mini prepping according to the
instructions provided in the Qiagen mini prep kit. The DNA samples from all three were sent out
to be sequenced to ensure that no mutations occurred and to verify that the construct was exactly
correct. In the meanwhile, all three samples were used to generate mRNA that could later be
injected into embryos should they prove to be correct. Mini prepped samples of the construct
were used to create mRNA using Ambion MEGAscript kit under RNAse-free conditions. Each
completed mRNA synthesis was tested for correct length and successful production of mRNA
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using gel electrophoresis. As demonstrated in Figure 4, all three reactions were successful. The
concentration and purity of mRNA in each sample was determined using spectrophotometry at
260 and 280nm. Then, these were diluted and prepared for the microinjection needles.
Figure 4: Gel Electrophoresis of mRNA production
Preparation of Microinjection Needles: The microinjection needles were prepared on
site using small glass tubes and a needle pulling apparatus. Then a small drop of dilute mRNA
with a small amount of glycerin was applied to the top of the needle and allowed to enter the
needle through capillary action. Microinjection solutions contained 2 mM mRNA and 20%
glycerol. Filled needles were stored at - 20˚C until use to prevent decomposition of the mRNA.
Animals: Lytechinus variegatus sea urchins were obtained from the North Carolina coast
(Duke Marine Lab). Spawning was induced by intracoelomic injection with 0.5 M KCl. Sperm
were kept in ice until use or were stored at 4˚C for up to several weeks until needed. Eggs were
used immediately after collection. The eggs were collected and cultured in artificial seawater at
23˚C7.
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Both eggs and sperm were examined under a microscope prior to use to ensure both were
in good condition. To prepare eggs for injection, they were arranged in injection dishes via
rowing. Eggs were rowed on dishes coated with protamine sulfate to cause the eggs to stick to
the dish. Rowing was performed by mouth pipetting eggs and depositing them in approximately
straight, single file lines on the dish. The eggs were injected within 5 minutes of fertilization
with 45-65 pg Alx1.GR.GFP mRNA solution using a Picopritzer II microinjection apparatus
(General Valve Corporation).
Confocal Microscopy: Once the embryos had reached the two-cell stage, about 1 hour
into normal development, they were analyzed using confocal microscopy, which allows the
embryos to be viewed in different planes of vision. The confocal microscope uses point
illumination and a pinhole in an optically conjugate plane to eliminate any image that is out of
focus. Because only one point is viewed at a time, the complete image must be reconstructed
from a scan over the desired image. The apparatus could also be set to view fluorescence due to
green florescent protein present in the embryo.
The10 μM dexamethasone solution was prepared in ethanol and a few drops of solution
were added to eggs at the two-cell stage. The embryos were incubated with the hormone for 45
min prior to a second confocal microscopy examination. Two focal planes of 2, 4 and 8-cell
stage embryos were analyzed under fluorescent light. In one plane of vision, the view was of
cytoplasm and the nucleus was seen as a darker area behind the plane of vision. The other plane
allowed for a view of inside the nucleus. Fluorescing embryos were examined to determine if
GFP was in the nucleus or the cytoplasm before hormone addition and again afterwards. Images
were taken in focal planes best displaying the location of the fusion protein.
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Optimization of the System: Alx1 expression does not begin naturally until the hatched
blastula stage. This causes several complications, including that the hatched blastula is free
swimming, making it difficult for image capture, and the nuclei are less visible because the cells
are smaller and more numerous at this stage. Therefore, prior to studying embryos injected with
Alx1.GR.GFP mRNA, the system was optimized using mRNA from a construct that had βCatenin in place of Alx1. For this control construct, the β-Catenin sequence was also ligated to
the GR ligand binding domain and green fluorescent protein genes to produce βCatenin.GR.GFP DNA. The upstream network component, β-Catenin, is expressed at the twocell stage and is naturally nuclear. These aspects made β-Catenin ideal to work with because the
embryos could be examined at early stages with fewer, larger cells. Also a construct of βCatenin.GR.GFP was already available and, therefore, could be used to optimize the system
while Alx1.GR.GFP was still being constructed. First, mRNA was produced from this construct
as previously described. Likewise, eggs were injected as previously described with the βCatenin.GR.GFP construct to test the system in sea urchin embryos. The presence of β-Catenin
in the cytoplasm prior to hormone addition would demonstrate that the glucocorticoid receptor
can exclude proteins from the nucleus, since β-Catenin is naturally nuclear by the 2-cell stage.
Also, if this protein is shown to migrate to the nucleus upon addition of hormone, then the
system is feasible in sea urchins. Because the β-Catenin system can be tested within a few hours,
it is easier to use in determining the appropriate amount of hormone to add and how long the
embryos should be incubated with the hormone.
Results:
System Optimization: Initially, the experiments using the dexamethasone system were
performed with β-catenin as a control. Optimization with the β-catenin construct showed that the
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construct is sequestered in the cytoplasm prior to hormone addition and enters the nucleus
rapidly upon dexamethasone addition. Embryos were first viewed using confocal microscopy at
the 2 and 4 cell stages. At this point, every embryo developing normally, as explained below,
and showing fluorescence had the green fluorescent protein in the cytoplasm only. Embryos that
were either not fluorescing or not developing normally were not examined for results. Because
fertilized eggs develop rapidly and become difficult to penetrate with a needle soon after
fertilization, not every egg on the plate is injected. Also, it is difficult to row eggs such that they
are single file, therefore only the eggs on the outside of the row are accessible to the needle. It is
normal for a large fraction of the eggs on the plate to go uninjected. Florescence was used as an
indication of successful injection. Also, injection may disrupt the fertilized egg such that it does
not develop normally. “Normal” embryos are considered to be those developing on the correct
time scale and that have cells that are approximately round.
The cells were viewed prior to hormone addition at a point where some embryos were at
the 2-cell stage, but some had already made it to the 4-cell stage. This timing made it so that we
could compare 4-cell stage embryos prior to hormone addition to 4-cell stage embryos after
hormone addition. Otherwise, it could be thought that the protein migrated to the nucleus
between the 2 and 4-cell stages as the result of something other than the hormone.
Figure 5 shows a 4-cell stage embryo prior to the addition of hormone. Note that the
entire embryo is glowing green. This is due to both baseline fluorescence, due to a small amount
of autofluorescence in urchin embryos and reflection from the green florescent protein.
Embryos not expressing GFP will still appear faintly green under the confocal. The actual green
florescent protein is only located in the very bright green halos surrounding each of the four
nuclei. The nuclei are the dark circular areas near the outside edge of each of the four cells.
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Figure 5: Confocal image of 4-cell stage prior to dexamethasone addition. The cell in the top
right corner has dotted white lines to indicate the location of the construct. The nucleus is the
dark area encircled by the GFP-containing fusion protein.
Figure 6 shows a different embryo at the 4-cell stage, but after the embryo had been
incubated with dexamethasone hormone. It is not as clear that this embryo is at the four cell
stage due to the angle at which the image was taken. The fourth cell is on the left side of the
image and is mostly behind the middle two cells. Note that now the areas of very bright green
are in the circles located towards the outside edge of each cell, indicating that the protein is now
inside the nucleus. Because the protein was perinuclear prior to transport, it could be argued that
this image still shows the protein around the nucleus and not in it, but viewed from above the
nucleus. Several images were taken in different focal planes to ensure that this is not the case
and the protein is in fact inside the nucleus. We also scanned to a level at which the nucleus of
the fourth cell could be viewed, and it too contained the protein in the nucleus and not around it.
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Figure 6: Confocal image of 4-cell stage after 45 min incubation with 10µM dexamethasone.
The nucleus of the bottom-most cell is indicated with an arrow. Note that the nucleus is now
fluorescing with the most intensity and is where the construct is located.
It was also important during these optimization experiments to ensure that once the βCatenin.GR.GFP fusion protein entered the nucleus, it remained there. Otherwise, the system
would not allow for the level of controlled expression needed. Therefore, we also took images of
an 8 cell-stage embryo roughly two hours later. These 8-cell embryos were from the same batch
as the 4-cell embryos pictured in Figures 5 and 6, and therefore underwent nuclear localization of
the fusion protein at the same time. As seen in Figures 7 and 8, which represent two halves of
the same embryo, the protein remains in the nucleus even after the embryo has further divided.
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Figure 7: Confocal Image of the top view of an 8-cell embryo
after 45 min incubation with 10µM dexamethasone
Figure 8: Confocal Image of the bottom view of the same 8-cell
embryo after 45 min incubation with 10µM dexamethasone
Alx1 Hormone-Inducible System: Trials with the Alx1 construct were equally
successful. In fact, the migration of the protein from the cytoplasm to the nucleus proved to be
even more rapid than previously estimated. In the β-Catenin.GR.GFP fusion protein trial, we
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allowed the hormone to incubate for 45 minutes before observing the embryos again under the
microscope. Because this worked so well, we checked on the embryos injected with the
Alx1.GR.GFP construct after only 20 minutes and found that migration had already occurred.
Figures 9 and 10 demonstrate this. Both are courtesy of Margarida Anjos, Carnegie Mellon
University.
Figure 9: Confocal image taken just prior to the addition of dexamethasone
Figure 10: Confocal Image taken 20 minutes after incubation with 10 µM dexamethasone
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Discussion: In an effort to analyze Alx1 function, a hormone inducible system that would allow
for temporal control of Alx1 activity was tested. Migration of the fusion protein to the nucleus is
rapid, persistent and appeared to occur in all embryos showing fluorescence. The speed at which
the fusion protein migrated from the cytoplasm to the nucleus was even faster than for the frog
system, which required a minimum of 2 hours of hormone treatment8. Because nuclear import
occurred within 20 minutes, the system will allow for very tight control of Alx1 expression in
future experiments. One could pick the exact stage desired for initiation of Alx1 expression
because the rapid import would allow for such precision. Persistence is also a key attribute for
the system because the system would not be useful if the construct did not remain in the nucleus.
The results demonstrate that even after the cells divide, the fusion protein remains nuclear, so
experiments can be performed with the assumption that Alx1 will continue to exert its function
several stages later if it is meant to. It is also important that successful nuclear transport occurred
in all embryos expressing GFP. Otherwise it would be very difficult to study the effects of Alx1
expression in a population of embryos because one would not be certain that all of them had
nuclear, and therefore active, Alx1. All of these features are essential for the hormone-inducible
expression system to be useful.
However, even though we have shown that the system works in principle, there is still
much to be done. First of all, the previously mentioned tests to ensure that the system is feasible
should be repeated because the samples sizes these assumptions are made on were very small.
Few eggs on the dish develop normally under the given conditions because the embryos prefer
natural seawater and the process of rowing the eggs on the Petri dishes sometimes damages
them. In addition, few eggs successfully acquire the construct and produce fusion protein. As
previously mentioned, it is very difficult to inject all or even most of the eggs on the plate. It
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helps to begin with a single file row, but only one who is very experienced with rowing can
achieve this. Also, eggs must be injected within about 5 minutes of fertilization or else a
fertilization envelop, which is difficult to penetrate, prevents injection. When there are hundreds
of eggs on the plate, it is very difficult to get to all of them within the allotted time. Injection can
also disrupt the embryo. Therefore, while the current results are very promising and consistent
for those embryos that both received the construct and developed normally, the system should
still be tested again with a larger sample size.
It also would be helpful to take videos of the construct actually migrating from the
cytoplasm to the nucleus instead of taking only confocal images. This way, it would be even
more certain that migration is occurring because of the addition of hormone, because one would
be able to see it happening. Also, we would be able to have a better idea of how long it takes for
migration to occur once the dexamethasone is added. The better the estimate we have of this
time frame, the more precisely we should be able to work with the temporally controlled system.
Knowing how long migration actually takes would also give us an idea of how rapid and
efficient the system is.
Once we have the system optimized, we still need to determine the effect of temporally
controlled expression of Alx1 using the hormone-inducible system on skeletogenesis.
Although the system could theoretically be used to analyze the temporally controlled expression
of many different genes of interest now that it is known that the system works in sea urchins, the
main purpose of implementing the system was to study Alx1. The system will be used to
determine the effects of over-expression of Alx1 both at the point in development when Alx1
naturally exerts its effect and at later points in development.
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Acknowledgements: I would like to thank Dr. Charles A. Ettensohn of Carnegie Mellon
University, Department of Biological Sciences for his intellectual guidance throughout the
project. I would also like to acknowledge Jennifer Leonard for her technological assistance and
stimulating conversation as well as Margarida Anjos for sharing her confocal images. These
individuals were invaluable sources of knowledge in all aspects of this project. I am also
grateful for my readers, Dr. Ildiko Kovach and Dr. Ann Corsi, for their useful criticism and
comments. This work was supported by the National Science Foundation Research Experiences
for Undergraduates Program sponsored by Carnegie Mellon University in 2007.
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