Transcript COULD β-CATENIN BE INVOLVED? - York College of Pennsylvania

Lithium Chloride Induces Exogastrulation and Teratogenesis in Developing
Sea Urchin (Lytechinus variegatus) Embryos: COULD β-CATENIN BE INVOLVED?
Cody Blymire
Department of Biological Sciences, York College of Pennsylvania, York, PA 17405
Project Summary:
Literature Review:
1McGrath
et al. (1991):
They noted that embryos exposed to LiCl demonstrated both a
delay in development and an increase in exogastrulation in a dose
dependent manner.
Increase LiCl, increase ß-catenin
The sea urchin is a very popular organism for scientific investigations. For this
research project, Lytechinus variegatus, was studied in regards to the toxic effects of
LiCl in delaying embryo development, causing exogastrulation, and affecting βcatenin expression during the gastrulation stage. Gastrulation is an extremely
important developmental stage in the sea urchin, and drastic changes in cell
arrangements occur. LiCl inhibits this developmental stage by causing the urchin
to not form a gut tube or archenteron (Figure 1B). This phenomenon implies an
effect on the cell-cell interactions and arrangements necessary for gastrulation to
occur properly. β-catenin is a multifunctional protein that is part of the WNT
signaling pathway that assists in cell-cell adhesion and cell fate determination. βcatenin expression is reported to be most abundant during sea urchin gastrulation,
and accumulation is most abundant around the developing archenteron. LiCl is a
well known vegetalizing agent, and has been demonstrated to delay development in
various organisms. LiCl was presented at various concentrations of 0 mM, 15 mM,
30 mM, and 60 mM, and the sea urchins embryos were placed in these
concentrations at the two-cell stage and allowed to develop. Morphological as well
as developmental observations were made. Results showed that LiCl inhibited the
development of embryos in a dose dependent manner in that the higher
concentration the more inhibition was observed. Morphological dysfunctions
included embryo fragmentations as well as exogastrulation. Due to lack of βcatenin antibody availability an ELISA test will be proposed and it is hypothesized
that as the concentration of LiCl increases, the expression of β-catenin would
increase as well in a dose dependent manner. This would imply that β-catenin may
play a vital role in sea urchin gastrulation.
Anticipated Results:
Research Design:
Sperm
Eggs
Expose and Fertilize
Urchin Gametes
2Miller
et al. (1995):
They researched that β-catenin modulates cell-cell adhesion during
gastrulation in the sea urchin.
They found that treating sea urchin embryos with LiCl enhanced
the level of β-catenin mRNA detected through Northern Blot
analysis.
They also noted that embryos treated with LiCl displayed an
increase in endoderm and mesoderm cells.
Figure 3: The anticipated results of the ELISA test in regards to
B-catenin expression and LiCl concentrations
Allow Embryos to Develop
to the 2-4 Cell Stage
2-4 cell
3Miller
and McClay (1997):
They determined that β-catenin is expressed at its highest level
during the gastrulation stage.
4Logan
et al. (1999):
They noted that when the archenteron is forming, β-catenin
expression is localized to cells forming the archenteron.
Introduction:
Gastrulation is a very important developmental occurrence
in sea urchins. During this stage, many cell-cell interactions
and cell relocations occur. It is during this stage that the
urchin embryo forms what is termed the archenteron or gut
tube (Figure 2). When sea urchin embyros are exposed to
LiCl they do not form an archenteron, rather they
exogastrulate. Urchins exposed to LiCl also demonstrate
enhanced expression of β-catenin mRNA. This β-catenin
may play a major role in signaling cells to successfully
develop the archenteron. This is the question that my
proposal aims to address, could β-catenin protein expression
be enhanced by LiCl exposure, thus potentially be involved
in causing this exogastrulation occurrence.
~2000 embryos per beaker of seawater ± LiCl
Add Embryos to Each
Corresponding Beaker
5Kitazawa
and Amemiya (2001) :
They studied the effects of LiCl on developing sea urchin embryos
(Peronella japonica).
They found that the developing archenteron was severely hindered
due to exposure to LiCl at various concentrations.
6Weitzel
et al. (2003):
They observed that over-expression of β-catenin hinders primary
mesenchyme cell (PMC) production.
They deterrmined that β-catenin is a powerful activator in the PMC
linage during the gastrulation stage.
0 mM
15 mM
30 mM
60 mM
Figure 4: The anticipated results of the ELISA spectrophotometer
reading values plotted against the varying LiCl treatments.
Continuous Exposure Through Gastrulation Stage
Once Embryos Reach
Gastrulation Stage,
Obtain Protein Lysates
Hypothesis:
As LiCl concentrations increase, the expression of
β-catenin protein will increase in a
dose dependent manner
Regular Gastrulation
Exo-gastrulation
0
B
A
15
30
60
Simulated Microtiter Wells
Total Protein Lysates From
~100 Embryos Added to
Each Coated Well; 10
Replicates, Each in
Duplicate
ELISA TEST
Gut
Plate Reader
Figure 5: The expected standard curve results of varying B-catenin concentrations
and their absorbance readings from an absorbance spectrophotometer.
Literature Cited:
1.
McGrath, Catherine, Robert McIsaac, and Susan G. Ernst. 1991. Altered Cell Fate in LiCl-treated
Sea Urchin Embryos. Developmental Biology. 147: 445-450.
2.
Miller, Jeffrey, Scott E. Fraser, and David McClay. 1995. Dynamics of Thin Filopodia During Sea
Urchin Gastrulation. Development. 121: 2501-2511.
3.
Miller, Jeffrey and David R. McClay. 1997. Changes in the Pattern of Adhesion JunctionAssociated B-catenin Accompany Morphogenesis in the Sea Urchin Embryo. Developmental
Biology. 192: 310-322.
4.
Logan, Catriona Y., Jeffrey R. Miller, Michael J. Ferkowicz and David R. McClay. 1999. Nuclear
B-catenin is Required to Specify Vegetal Cell Fates in the Sea Urchin Embryo. Development.
126: 345-357.
5.
Kitazawa, Chisato and Sharon Amemiya. 2001. Regulating Potential In Development of a
Direct Developing Echinoid, Peronella japonica. Develop. Growth Differ. 43: 73-82
6.
Weitzel, Heather E., Michele R. Illies, Christine A. Byrum, Rongui Xu, Athula H. Wkramanayake,
and Charles A. Ettensohn. 2004. Differential Stability of B-catenin Along the Animal-Vegetal
Axis of the Sea Urchin Embryo Mediated By Dishevelled. Development. 131: 2947-2956.
Acknowledgements:
Figure 2: The gastrulation process in urchin embryos relies on
both PMC’s (primary mesenchyme cells) and SMC’s (secondary
mesenchyyme cells).
Figure 1: Regular gastrulating sea urchin embryo(A) vs. an exo-gastrulating sea urchin embryo
exposed to LiCl(B).
Absorbance
Spectrophotometer
.
• York College of Pennsylvania
• Dr. Ricker for her continuous advice and support,
you are the best!!
• Dr. Smith