The long-term goals of this work are to understand cellular and molecular mechanisms of endoderm development during vertebrate embryogenesis. Our current understanding of this process has benefited from studies in several model systems including Xenopus, zebrafish and mouse. Our work in zebrafish has identified three transcription factors important for endoderm formation, namely the Mix-type homeodomain factor Bon, the zinc finger factor Gata5 and the Sox-type factor Cas. We further assembled these and other factors into a genetic pathway of endoderm formation. We have recently completed a screen for additional mutations that affect the expression of sox17, an early endodermal marker. This screen has led to the identification of 9 mutants that we are currently recovering. Interestingly, one of these mutations affects the smoothened gene, thereby unexpectedly implicating Hedgehog (Hh) signaling in early endoderm formation. While Hh signaling has previously been implicated in endoderm development, all studies thus far have pointed to a late role for Hh in this process. Our initial results, however, indicate that Hh signaling plays a very early role in endoderm development.
We aim to continue our investigation of endoderm development in zebrafish by focusing on this new set of early endoderm mutants and propose the following specific aims: 1) recover and determine the phenotype of the 8 new endoderm mutants; 2) investigate the role of Smoothened/Hh signaling in endoderm formation. Detailed studies of embryos with blocked or enhanced Smoothened/Hh signaling will allow us to study the precise role of this signaling pathway in early endoderm differentiation and patterning; and 3) analyze in detail, including positionally cloning, two genes from our new set of mutants, s421 and s468, that regulate endoderm formation in a previously undocumented manner. These molecular genetic studies of endoderm formation will help provide the necessary context for further investigating the pathophysiology of malformations and malfunctions of the human gut and its associated organs, and may also facilitate differentiation-based strategies for treatment of a variety of disease states. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK058181-06
Application #
6923678
Study Section
Special Emphasis Panel (ZRG1-BDA-C (02))
Program Officer
Sato, Sheryl M
Project Start
2000-08-15
Project End
2008-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
6
Fiscal Year
2005
Total Cost
$303,000
Indirect Cost
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Yin, Chunyue; Kikuchi, Kazu; Hochgreb, Tatiana et al. (2010) Hand2 regulates extracellular matrix remodeling essential for gut-looping morphogenesis in zebrafish. Dev Cell 18:973-84
Aanstad, Pia; Santos, Nicole; Corbit, Kevin C et al. (2009) The extracellular domain of Smoothened regulates ciliary localization and is required for high-level Hh signaling. Curr Biol 19:1034-9
Anderson, Ryan M; Bosch, Justin A; Goll, Mary G et al. (2009) Loss of Dnmt1 catalytic activity reveals multiple roles for DNA methylation during pancreas development and regeneration. Dev Biol 334:213-23
Shin, Chong Hyun; Chung, Won-Suk; Hong, Sung-Kook et al. (2008) Multiple roles for Med12 in vertebrate endoderm development. Dev Biol 317:467-79
Trinh, L A; Meyer, Dirk; Stainier, Didier Y R (2003) The Mix family homeodomain gene bonnie and clyde functions with other components of the Nodal signaling pathway to regulate neural patterning in zebrafish. Development 130:4989-98
Kikuchi, Y; Agathon, A; Alexander, J et al. (2001) casanova encodes a novel Sox-related protein necessary and sufficient for early endoderm formation in zebrafish. Genes Dev 15:1493-505