This study examined the effects of inhibiting the Hippo signaling pathway during sea urchin embryogenesis. Researchers treated sea urchin embryos with verteporfin, an inhibitor of YAP which is a key component of the Hippo pathway. They found that inhibiting the Hippo pathway delayed cell cleavage and gastrulation. While marker genes for specific cell types were still expressed, the patterns of expression were altered. Inhibition of the Hippo pathway resulted in epithelial cells that were larger in size and fewer in number, suggesting YAP is involved in epithelial organization and proliferation during sea urchin development.

1. Analyzing gene expression following perturbations of the Hippo
signaling pathway during sea urchin embryogenesis
This research was supported by Susquehanna University Research Partners Program and
an S-STEM grant from the National Science Foundation.
Abstract
Cell differentiation and morphogenesis rely on cell-signaling pathways that convey key
information to cells about their roles during embryogenesis. We are studying the role of the
Hippo pathway in the sea urchin, Lytechinus pictus, using loss-of-function analysis with a small
molecule inhibitor. We are determining the effects of pathway inhibition on gene expression
through in situ hybridization of cell-type specific genes. We have previously shown that the PCP
pathway is required for invagination of the archenteron but certain endoderm and mesoderm
specific genes appear to be normally expressed4. We are extending those studies by examining
additional markers, specifically aa29, myosin, endo16, and msp130. We are examining changes in
the expression of these genes in embryos in which the Hippo pathway (required for cell
proliferation) has been disrupted. These studies will help link these signaling pathways to specific
events in cell differentiation in the sea urchin embryo.
Introduction
The Hippo pathway is an evolutionarily conserved pathway involved in the
regulation of cell-proliferation and apoptosis. The Hippo pathway has been
best studied in Drosophila. The Yes-associated protein (YAP) plays a key role
in the Hippo pathway, acting as a transcription factor to control cell
proliferation.1 YAP is translocated into the nucleus where it interacts with
TEAD1-4 and other such transcription factors to induce cell proliferation and
apoptotic inhibition genes. Activation of the Hippo pathway leads to
degradation of YAP, therefore reducing cell proliferation. Therefore, defects in
the regulation of the Hippo pathway can contribute to tumor development.
The Hippo pathway is composed of a kinase cascade, where Mst1/2 and Sav1
phosphorylate LATS1/2. LATS1/2 phosphorylates and inhibits the YAP
complex.1 The role of the Hippo pathway in the development of the sea urchin
has not been determined.
Questions
Results and Discussion
Material and Methods References
Acknowledgments
Alyssa Bolger, Justice Bufford, Sophia Francisco, Nina Ngo, Janeily Perez, and Margaret Peeler PhD
Department of Biology, Susquehanna University, Selinsgrove, PA 17870
Figure 3. Early developmental effects of verteporfin on sea urchin
(Lytechinus pictus) embryos. Embryos were treated with verteporfin or a
DMSO-only control 20 minutes post fertilization (PF). Embryos were counted
to determine the number of cells at 1 hour, 1.5 hours, and 2.5 hours PF.
Embryos were categorized as having 1-cell, 2-cell, 4-cell, 8-cell and 16-cells. Cell
counts were converted into percentages. The verteporfin treated embryos
exhibited developmental delays by 2.5 hours PF.
Sea Urchin Cultures
• Injected sea urchins with 0.5M KCl to spawn their eggs and sperm
• Extracted eggs and sperm from female and male sea urchins and fertilized the eggs with a dilute sperm suspension
• Cultured embryos in 2ug/ml of verteporfin (Sigma Aldrich) in DMSO, using equal volume of DMSO for controls
• Tracked and observed developmental effects the inhibitor had on the embryos for early development (1-3 hrs) and
further time periods of embryogenesis (20, 28 and 48 hrs)
In Situ Hybridization
• Embryos were fixed in 4% paraformaldehyde and post fixed in methanol
• Dig-labeled RNA probes were made from linearized plasmids using SP6 or T7 polymerases
(Retroscript kit, Thermo Fisher Scientific)
• Inhibited and control embryos were hybridized with probes at 55°C overnight and detected using alkaline
phosphatase staining as described in Long et al., 20154
We have chosen to use verteporfin to inhibit YAP, preventing it from entering
the nucleus and activating cell-proliferation. Specifically, we are examining
the effects of verteporfin on the early development of the sea urchin embryo,
including analysis of the expression of embryogenic associated genes, aa29
(encodes enzymes of the nucleotide metabolism), endo16 (encodes a large
extracellular protein expressed in the endoderm), msp130 (encodes a
130 kD cell-surface glycoprotein), and myosin (encodes for actin-based motor
proteins). These genes are expressed in a cell type specific manner and can
help determine the effect of YAP inhibition on the formation of these cells.
• What expression patterns should we expect to see for each gene? When and
where should we see them?
• Does verteporfin disrupt the expression pattern of each gene?
Figure 1. Signaling of the
Hippo pathway2. (a) When
the Hippo pathway is on, YAP
degrades and cannot trigger cell
proliferation and block
apoptosis. (b) When the Hippo
pathway is off, YAP enters the
nucleus and activates genes for
cell proliferation and block
apoptosis.
Figure 2. Quantitative Developmental Transcriptomes of S. purpuratus.
RNA Sequence of S. purpuratus YAP was used to determine when and where
YAP was turned on and off during sea urchin development. This figure shows
how many transcripts per embryo were produced over time. Levels of
transcription are high at fertilization, significantly decrease through ten hours,
and then increase.
1. Guan, K. (2014, July). Hippo Signaling Pathway. Cell Signal Technology.
2. Johnson, R., & Halder, G. (2013). The two faces of Hippo: Targeting the Hippo pathway for
regenerative medicine and cancer treatment. Nature Reviews Drug Discovery Nat Rev
Drug Discov, 13(1), 63-79. doi:10.1038/nrd4161
3. Warr, D., Jones I., and Peeler, M. (2016). Yes-associated protein inhibitor verteporfin impacts the
embryogenesis of the sea urchin. Senior Honors Thesis, Susquehanna University.
4. Long et al., 2015. JNK is required for invagination but not differentiation of the sea urchin
archenteron. genesis 53:762-769
VerteporfinControl
A B C
D E F
24 Hours 28 Hours 48 Hours
Figure 5. In situ hybridization of secondary mesenchymal cells using the aa29
gene performed at the 24 hour, 28 hour, and 48 hour mark of L. pictus
development. (A-C) The control embryos displayed a greater number, as well as a
greater dispersal, of secondary mesenchyme cells. (D-F) The inhibited embryos
demonstrated more clumping than the control embryos, especially near the tip of
the archenteron and appear to have fewer positive staining cells.
Figure 6. In situ hybridization of the cells of the gut using the endo16 gene
performed at the 24 hour, 28 hour, and 48 hour mark of L. pictus
development. All embryos, control and inhibited, expressed staining of the
invaginating archenteron, but inhibited embryos were delayed. The mouth had not
formed by 48 hours in treated embryos.
Figure 7. In situ hybridization of primary mesenchymal cells using the msp130
gene performed at 24 hour, 28 hour, and 48 hour mark of L. Pictus
development. The control (A, B) and inhibited (D, E) embryos both looked very
similar at the 24 hour and 28 hour mark, with the stained clumps of PMCs near the
vegetal plate and in the blastocoel. (C) The control embryos at 48 hours displayed
staining of the PMCs in ventral lateral clusters. (F) The inhibited embryos at 48
hours showed an abnormal staining pattern of PMCs.
Figure 8. In situ hybridization of the muscle cells surrounding the archenteron
using the myosin gene performed at the 24 hour, 28 hour, and 48 hour mark of
L. pictus development. The control (A, B) and inhibited (D, E) embryos at 24 and 28
hours did not show any staining, as expected. (C) At 48 hours, the control embryos
displayed clear staining of the muscles surrounding the developing archenteron. The
embryos developed normally, and were in the prism stage of development. (F) The
inhibited embryos at 48 hours were delayed, but they still showed staining of the
muscle cells surrounding the archenteron.
VerteporfinControl
24 Hours 28 Hours 48 Hours
VerteporfinControl
24 Hours 28 Hours 48 Hours
VerteporfinControl
24 Hours 28 Hours 48 Hours
Figure 4. Early morphological
effects of sea urchin cells treated
with verteporfin (Lytechinus
pictus). These cells exhibited
delayed cell division, but developed
normally during early cleavage.
• YAP inhibition delays cell cleavage and gastrulation
• Although YAP inhibited embryos express aa29, endo16, msp130 and myosin,
the pattern of stained cells is altered for all but myosin
• YAP may be involved with the epithelium integrity and its overall
organization. Epithelial cells appear larger and reduced in number (due to
fewer cell divisions) in inhibited embryos
Conclusion
Figure 9. The effects of verteporfin on epithelial cell
division and cell size in L. pictus after 17 hours.
(A) The control embryos exhibited a larger quantity of
epithelial cells, due to more cell division. (B) The
inhibited embryos demonstrated larger and fewer
epithelial cells with more defined cell borders.1