Download The characterization of heart progenitor cell

HEART PROGENITOR CELL MOVEMENT IN DEVELOPING FROG EMBRYOS
Timothy Simon1, Tim Jackson1, Lance Davidson1,2
Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15260
2
Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA 15260
1
INTRODUCTION
Congenital heart defects (CHD) include some of the
most prevalent birth defects in the US, affecting nearly
40,000 infants each year [1]. CHD is the incomplete or
abnormal formation of the heart or intrathoracic vessels
leading to a significant effect on the subject’s functionality
[2]. While the causes of CHD are poorly understood in
humans, much effort has been placed in understanding the
molecular causes of CHD in model organisms. For instance,
missense mutations in phosphatase proteins, specifically
SHP-2, can cause this structural irregularity during the
formation of the heart in the developing frog embryo [3]. In
this study, the specific mutation SHP-2N308D will be explored
extensively
Figure 1: Schematic of normal SHP-2 protein and mutation
constructs, including SHP-2N308D [3]
Y27632 on a frog embryo through the analysis of time lapse
movies. We intend to calculate the area of the heart region,
comparing across several varying concentrations of Y27632.
In order to understand the mechanics of heart progenitor cell
migration, we will track the heart progenitor cells and
determine the strains within the tissues they travel through.
We must also calculate principal strain on the epithelial layer
determining whether it contracts or expands during the
period of progenitor cell migration.
MATERIALS AND METHODS
To visualize cell movements within the embryo we must
stabilize the tissues through which heart progenitors migrate.
We mount the embryos rigidly on their dorsal surface so that
we can follow cell movements on the embryo's ventral
surface where heart progenitors migrate and the two clusters
of cells join to form the heart. First, we remove the vitelline
membrane of post-gastrulation and pre-neurulation Xenopus
laevis embryos to allow for surgical manipulation.
Microsurgery is then performed on the post-gastrula embryos
(stage 12.5). Small incisions are made along the dorsal side
of the embryo. These incisions will expose the deep cells
Behind the incomplete migration of the heart progenitor
cells is the mutated SHP-2N308D pathway. The mutant
phosphatase protein SHP-2N308D induces hyper-activation of
the Rho-associated coiled protein kinase (ROCK). Changes
in ROCK activity will alter the stiffness of the tissue through
the reorganization of the Xenopus Laevis cytoskeletal tissue.
As the cytoskeletal structure is altered and the tissue stiffness
increases, the heart progenitor cells are unable to migrate
from the lateral plate mesoderm to the ventral midline and
fuse to form a complete, fully functional heart [3].
The pharmacological compound, Y27632, inhibits
ROCK activity. The complete effect that this compound has
on the development on the SHP-2 mutated embryo and the
normal, non-mutated embryo has not been fully characterized
[3]. However, no connection has been made between the
inhibition of ROCK and the rescue of the mutated SHP2N308D pathway in the Xenopus Laevis embryo.
It was hypothesized that the introduction of Y27632 to a
normally developing embryo will cause abnormal formation
of the heart thus proving that compound Y27632 is capable
of inhibiting ROCK hyperactivity and rescuing SHP-2
mutated embryos. To substantiate this hypothesis, we must
characterize the effect of ROCK-inhibiting compound
beneath the epithelial layer.
Figure 2: Diagram of the procedure described above. The
red X indicates the position of the small incisions made after
de-vitellinizing the embryo. [4]
Embryos are plated, incisions down, onto a fibronectincoated dish and treated with varying concentrations of the
compound Y27632. 24 hour time-lapse movies centered on
the heart-forming region on the ventral side of the embryo
were collected. These images were processed and analyzed
in ImageJ. The area of the heart-beating region was
normalized then calculated. A principal strain map of the
epithelial layer, detailing the specific areas of contraction and
expansion, was also created using ImageJ software. The
embryo was then fixed and stained and imaged using a laser
scanning confocal microscope (Leica SP5).
1
Area of Heartbeat (mm2)
RESULTS
After treating the embryos with a solution of 50 μM
Y27632 and collecting a time lapse video of their
development they were fixed and stained for fibrillin.
Confocal stacks were maximally projected into a single
image. A noticeable difference was evident upon comparing
the Y27632 treated embryo and the embryo left in control
media. The Y27632-treated embryo’s heart formed a bifid
heart or incompletely fused heart. The control embryo’s heart
progenitor cells successfully merged. Because the Y27632
inhibits ROCK, the heart progenitor cells were incapable of
migrating all the way to the ventral midline and fusing
together fully.
We retrospectively identified the regions of the embryo
contributing to the heart in long term time-lapse sequences
that culminate in the beating heart. Time-lapse images were
analyzed using ImageJ software to calculate the area of the
heart-beating region in the embryos. Once the general area is
determined through a t-projection image, the precise area is
thresholded to accurately calculate the pixel area where the
heart cells contract. The pixel area can they be converted to
square mm. This procedure was done for embryos treated
with 50 μM Y27632 and the control with 0 μM Y27632.
emerges of enabling successful heart progenitor cell
migration in a previously mutated Xenopus Laevis frog
embryo.
In the future, the connection between congenital heart
disease and the hyper-activity of ROCK must be solidified.
In order to conclusively determine that lessening ROCK
hyper-activity with Y27632 will rescue the mutated SHP-2
pathway and allow for complete heart formation, the
molecular and cellular mechanics behind heart migration
must be explored further. It must also be effectively
confirmed that the hyper-activity of ROCK is in fact
downstream of the SHP-2N308D mutant and will allow for the
normal formation of the heart.
Preliminary data suggests that Y27632-induced ROCK
inhibition prevents correct fusion of the bilateral heart
progenitor cell populations. However, in order to make a
statistically
significant
conclusion
regarding
the
hypothesized outcome, more experiments and data is needed.
The creation of a cardiogenic fate map is another tool that
can be utilized to understand better the mechanics of heart
progenitor cell movement. Using DiI, a fluorescent dye that
localizes in the cell membrane, heart-forming cells can be
tracked from the early stages of the Xenopus Laevis embryo
up until the beginning of heart contraction. The far-reaching
goal of this project is to relate the development of the
Xenopus Laevis heart to the human heart and thus be able to
better understand the causes of Congenital Heart Disease.
This long-term goal is far off in the future, however,
understanding of the mechanics of heart progenitor cell
migration is needed to reach it.
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50 μM
50 μM
0 μM
ACKNOWLEDGMENTS
I would like to acknowledge Tim Jackson and Dr. Lance
Davidson for their help throughout. I would also like to thank
Jesse Lowe and Dr. Borovetz for helping me through the
entire process this semester.
0 μM
Concentration of Y27632
Figure 2: Graph
concentrations
of
heartbeat
areas
and
solution
REFERENCES
1. Hoffman, J. I. E., Kaplan, S. The Incidence of
Congenital Heart Disease. JACC.
2002;39(12):1890-1900.
2. Mitchel, S. C. et al. Congenital Heart Disease in
Births. 1971;43(3):323-32.
3. Langdon, Y. et al. SHP-2 acts via ROCK to regulate
the cardiac actin cytoskeleton. 2012; 139(5):94857.
4. Drawings after Nieuwkoop and Faber.(1967)
Normal Tables of Xenopus laevis (Daudin).
Amsterdam: Elsevier North-Holland Biomedical.
A previously made algorithm that utilized Insight
Toolkit (ITK) was used to calculate the principal strain on
the epithelial layer and create a tracking grid on the epithelial
cells as the heart progenitor cells migrated. The algorithm
displays contraction and expansion of the epithelial layer of
the developing embryo.
DISCUSSION
Confocal imagery of the Y27632-treated embryo
shows the incomplete fusion of the heart progenitor cells
from the opposite lateral plate mesoderm. The treated
embryo forms a bifid heart . The contractile area of the
embryo treated with the compound is larger than that of the
embryo treated with the control carrier, DMSO. The larger
area in the Y27632 treated cells attests to the partial
migration and merging of the heart progenitor cells as the
Xenopus Laevis embryo develops. The abnormal heart
formation in the treated embryo indicates the inhibition of
ROCK. If ROCK hyper-activity could be inhibited or
diminished in a SHP-2 mutant frog embryo, the possibility
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