Dorsal-ventral polarity in the Drosophila embryo is initiated during oogenesis by expression of the Pipe sulfotransferase in the ventral follicle cells which, by an unknown mechanism, leads to the localized activation of a serine protease cascade in the perivitelline space between the embryonic membrane and the eggshell. Our long-term goal is to understand how this serine protease cascade is spatially regulated. The objectives of this application are to elucidate the mechanism through which spatial restriction of the serine protease cascade is implemented and to identify the target of Pipe action in the follicle cell layer. Our central hypothesis is that Pipe activity in the ventral follicle cells results in the sulfation of a glycoprotein or glycolipid that is secreted into and localized within the ventral perivitelline space where it brings about the localized activation of the serine protease cascade. The rationale for the proposed research is that it will greatly expand our understanding of DV pattern formation and will also provide insight into mechanisms that regulate analogous localized serine proteolytic events that influence human health such as the ones involved in the formation and breakdown of blood clots. Blood coagulation and fibrinolysis are critical factors in the etiology of thrombotic diseases such as heart attack and stroke which represent leading causes of death worldwide. Thus, the proposed research is relevant to the mission of the NIH to obtain fundamental knowledge that will potentially reduce the burden of disease. We will pursue two Specific Aims: 1) To determine the extent to which the activity of the serine protease cascade is regulated by the localization of the proteases themselves, by their spatially restricted activation or activities, or by physical interactions with one another that modulate their activities. Functional, tagged versions of the proteases will be used to examine their processing and spatial distribution and to investigate protein-protein interactions in wildtype and various genetic backgrounds. 2) To identify genes involved in the synthesis of the target of Pipe sulfotransferase activity in the ventral follicle cells. Genetic screens will be carried out to identify mutations that lead to the production of dorsalized embryos by mutant females. The proposed research is significant as it will yield new insights into mechanisms that regulate serine protease action and thus may contribute to the development of improved therapeutic agents for the modulation of serine proteases that influence human health. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Development - 2 Study Section (DEV2)
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Haynes, Susan R
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University of Texas Austin
Schools of Arts and Sciences
United States
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Amarnath, Smita; Stevens, Leslie M; Stein, David S (2017) Reconstitution of Torso signaling in cultured cells suggests a role for both Trunk and Torso-like in receptor activation. Development 144:677-686
Stein, David S; Stevens, Leslie M (2014) Maternal control of the Drosophila dorsal-ventral body axis. Wiley Interdiscip Rev Dev Biol 3:301-30
Stein, David; Cho, Yong Suk; Stevens, Leslie M (2013) Localized serine protease activity and the establishment of Drosophila embryonic dorsoventral polarity. Fly (Austin) 7:161-7
Cho, Yong Suk; Stevens, Leslie M; Sieverman, Kathryn J et al. (2012) A ventrally localized protease in the Drosophila egg controls embryo dorsoventral polarity. Curr Biol 22:1013-8
Cho, Yong Suk; Stevens, Leslie M; Stein, David (2010) Pipe-dependent ventral processing of Easter by Snake is the defining step in Drosophila embryo DV axis formation. Curr Biol 20:1133-7
Zhang, Zhenyu; Zhu, Xianjun; Stevens, Leslie M et al. (2009) Distinct functional specificities are associated with protein isoforms encoded by the Drosophila dorsal-ventral patterning gene pipe. Development 136:2779-89
Zhang, Zhenyu; Stevens, Leslie M; Stein, David (2009) Sulfation of eggshell components by Pipe defines dorsal-ventral polarity in the Drosophila embryo. Curr Biol 19:1200-5
Stein, David; Cho, Yong Suk; Zhang, Zhenyu et al. (2008) No requirement for localized Nudel protein expression in Drosophila embryonic axis determination. Fly (Austin) 2:220-8