THE ROLE OF YES-ASSOCIATED PROTEIN IN SEA URCHIN EMBRYOGENESIS: A LOSS-OF-FUNCTION ANALYSIS DAVID AURAND,  ACARA CARTER, BEATRIZ FUENTES-MONTESINO,  ALISSA MARCHIONE, NINA NGO, BEN WEISSTIFFANY BEINLICH, CASSANDRA DESTEFANO, NOAH DIAZ-PORTALATIN, KACIE MINIUM, IMANI SMITH, SARAH TALABISKADR. MARGARET PEELER, PHD

THE HIPPO/YAP PATHWAY BACKGROUND AND IMPORTANCE

(Halder and Johnson, 2011)

• Hippo regulates cell proliferation and apoptosis

• Yes-associated protein (YAP) as transcriptional co-activator

• Important for regulation of tissue homeostasis

• Dysregulation of this pathway affects organ development and can cause cancer in humans

COMPONENTS OF THE HIPPO PATHWAY EMPHASIS OF CELL PROLIFERATION AND BLOCKING APOPTOSIS

• When Hippo signaling is active a protein kinase cascade prevents YAP from entering the nucleus

• When Hippo signaling is inactive YAP activates transcription

• When transcription is active YAP activates genes which result in increased cell proliferation and anti-apoptosis signaling

HIPPO/YAP FUNCTION IN EMBRYOGENESIS

• Knockout of Sav1, Mst1/2, or Lats 1/2 kinases in cardiomyocytes during embryonic development in mice

• In Drosophila deletions of Wts leads to uncontrolled cell proliferation

• The Hippo pathway is not as well characterized in sea urchins but provides a good model organism.

A HIGHLY CONSERVED PATHWAY

YAP HOMOLOG STRONGYLOCENTROTUS PURPURATUS FOUND WITHIN OTHER SPECIES

THE SEA URCHIN: A BIOMEDICAL RESEARCH MODEL

• Sequenced genome in 2006• Strongylocentrotus Purpuratus

• Echinoderms• Free living, motile invertebrates• Closest known relative to the chordates

• In lab• Efficient • Translucent 

• In recent studies• Fertilization, cell division and

gastrulationSodergren, E., et al., 2006

Hilman, D., & Gat, U., 2011

Question:  What role does YAP play in regulating cell division during embryogenesis

in sea urchins?

YAP TRANSCRIPTOME EXPRESSION THROUGHOUT EMBRYOGENESIS

Hypothesis: If YAP is inhibited, then the rate of cell proliferation will   decrease or not

occur at all. 

MATERIALS AND METHODS 

Loss of function analysis using YAP specific inhibitor verteporfin

Verteporfin blocks YAP in developing sea urchin embryos 

Used different concentrations of verteporfin to observe effects: 0ug/mL, 1ug/mL, 2ug/mL and 5ug/mL

• At 24hrs PMC ingression and archenteron invagination had occurred

• At 48hrs control embryos were at the pluteus stage with greater archenteron formation

0 ug/mL 1 ug/mL 2 ug/mL 5 ug/mL

YAP HAS MINOR ROLE ON EARLY CLEAVAGE STAGES 

PROLIFERATION BETWEEN 8-18 HOURS CORRESPOND WITH YAP EXPRESSION RATE

8 Hours10 Hours

18 Hours

YAP INHIBITION ALTERS CELL NUMBER AT GASTRULA STAGE (24 HOURS)

YAP INHIBITION AFFECTS SKELETOGENIC CELL NUMBER AND DISTRIBUTION

VERTEPORFIN SLOWS EMBRYO GROWTH AT 48 HOURS  

YAP INHIBITION DOES NOT AFFECT MITOTIC CELL COUNT BUT ALTERS THEIR LOCALIZATION 

CONCLUSION 

• The use of Verteporfin has an effect from 4-8 hours and again between 24-48 hours, this trend is supported by the YAP transcriptome. 

•  YAP suppression has shown a significant effect on PMC and mitotic cell distribution.  

• The use of an inhibitor for YAP expression mimics activation of the Hippo pathway thus allowing a better understanding of the regulatory pathway in sea urchins during embryogenesis. 

FURTHER RESEARCH  

Apoptosis Assay EdU staining at earlier times  PMC and Archenteron staining after 48 hours Other inhibitors of the pathway   The inhibition of YAP affects formation and

placement of archenteron, is it affecting other tissues other than PMCS?

ACKNOWLEDGEMENTS

Dr. Margaret Peeler The other half of our team: 

Tiffany Beinlich, Cassandra Destefano, Noah Diaz-Portalatin, Kacie Minium, Imani Smith, Sarah Talabiska

Dr. David Matlaga and Dr. Alissa Packer for teaching us how to perform ANOVAs Department of Biology at Susquehanna University 

REFERENCES

Brodowska, K., Al-Moujahed, A., Marmalidou, A., Meyer zu Horste, M., Cichy, J., Miller, J.W., Gragoudas, E., and Vavvas, D.G. (2014). The clinically used photosensitizer Verteporfin (VP) inhibits YAP-TEAD and human retinoblastoma cell growth in vitro without light activation. Experimental Eye Research 124; 67-73  

Eagle, H., and Levine, E.M. (1967). Growth regulatory effects of cellular interactions. Nature 213;1102-1106   Decker, G.L. and Lennarz, W.J. (1988). Skeletogenesis in the sea urchin embryo. Development 103:231-247.    Gumbiner, B.M., and Kim, N. (2014). The Hippo-YAP signaling pathway and contact inhibition of growth. Journal of

Cell Science 127:709-717.   Halder, G., and Johnson, R.L. (2011). Hippo Signaling: growth control and beyond. Development 138: 9-22.    Harvey, K.F., Pfleger, C.M., and Hariharan, I.K. (2003). The Drosophila Mst ortholog, hippo, restricts growth and

cell proliferation and promotes apoptosis. Cell 114:457–467.     Harvey, K.F., Zhang, X., and Thomas, D.M. (2013). The Hippo Pathway and Human Cancer.   Nature Reviews-

Cancer 13: 246-257. Hiemer and Varelas, 2013   Sodergren, E., Weinstock, G. M., Davidson, E. H., Cameron, R. A., Gibbs, R. A., Angerer, R. C., ... &

Coffman, J. A. (2006). The genome of the sea urchin Strongylocentrotus purpuratus. Science, 314(5801), 941-952.

REFERENCES Hilman, D., and Gat, U. (2011). The Evolutionary History of YAP and the Hippo/YAP Pathway. Molecular Biology and Evolution 28: 2403-2417.  Hinegardner, R.T. (1967). Growth and development of the laboratory cultured sea urchin. Biol. Bull.,137: 465-475. Liu-Chittenden, Y., Huang, B., Shim, J.S., Chen, Q., Lee, S., Anders, R.A., Liu, J.O., and Pan, D. (2012). Genetic and pharmacological

disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP. Genes and Development 26:1300-1305.  Justice, R.W., Zilian, O., Woods, D.F., Noll, M., and Bryant, P.J. (1995). The Drosophila tumor suppressor gene warts encodes a homolog

of human mytonic dystrophy kinase and is required for the crontol of cell shape and proliferation. Gene & Development 9:534-546.  Nishioka, N., Inoue, K., Adachi, K., Kiyonari, H., Ota, M., Ralston, A., Yabuta, N., Hirahana, S., Stephenson, R.O., Ogonuki, N.,

Makita, R., Kurihara, H., Morin-Kensicki, E.M., Nojima, H., Rossant, J., Nakao, K., Niwa, H., and Sasaki, H. (2009). The Hippo signaling pathway components Lats and YAP pattern TEAD4 activity to distinguish mouse trophectoderm from inner cell mass. Developmental Cell 16: 398-410.

Tapon, N., Harvey, K.F., Bell, D.W., Wahrer, D.C.R., Schiripo, T.A., Haber, D.C.R., and Hariharan, I.K. (2002). Salvador promotes both cell cycle exit and apoptosis in Drosphila and is mutated in human cancer cell lines. Cell 110: 467-478.

Varelas, X. (2014). The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease.Development 141:1914-1626 Wang, C., Zhu, X., Feng, W., Yu, Y., Jeong, K., Guo, W., Lu, W., and Mills, G.B. (2016). American Journal of Cancer Research 6:27-37. Wei, X., Shimizu, T., and Lai, Z.C. (2007). Mob as tumor suppressor is activated by Hippo kinase for growth inhibition in Drosophila. The

EMBO Journal 26:1772- 1781.  Zhao, B., Wei, X., Li, W., Udan, R.S., Yang, Q., Kim, J., Xie, J., Ikenoue, T., Yu, J., Zheng, P., Li, L. Ye, K. Chinnaiyan, A., Halder, G.,

Lai, Z., and Guan, K. (2007). Inactivation of YAP oncoprotein by the Hippo Pathway is involved in cell contact inhibition and tissue growth control. Genes and Development 21:2747-2761.  

Zhao, B., Ye, X., Yu, J., Li, L., Li, W., Li, S. Yu. J., Lin, J.D., Wang, C., Chinnaiyan, A.M., Lai, Z., and Guan, K. (2008). TEAD mediates YAP-dependent gene induction and growth control. Genes and Development 22:1962-1971.  

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