Endocrine islet cell production solves several distinct yet related problems: endocrine commitment, cell type specification, islet cell differentiation, and maturation. In this grant application, we propose to utilize Myt1 gene activity manipulation and combinatorial cell marking to dissect these processes. High Ngn3 expression in the multipotent pancreatic progenitor cells is critical for islet cell differentiation. Because Ngn3 binds its own promoter to inhibit transcription, other activators are required to break the self-inhibitory loop for robust Ngn3 expression, which is essential for endocrine commitment. It is clear that Ngn3-independent mechanisms specify endocrine islet cell type. Yet the code that determines the eventual fate of each islet cell precursors remains unknown. Finally, newly differentiated endocrine cells fine-tune their gene expression pattern and cellular structure to give rise to mature functional islet cells. But the molecular mechanisms that coordinate the maturation process are unclear. Our studies over the previous funding period have uncovered several mechanisms that could potentially bridge some of the above gaps. First, we have demonstrated that Myt1 and Ngn3 form a feed-forward expression loop to promote neogenesis. This finding suggests that Myt1 could play an essential role in breaking the Ngn3 self-inhibitory loop to facilitate endocrine commitment. Second, inactivating Myt1 results in abnormal endocrine differentiation and function. Unexpectedly, losing Myt1 induces an unexpected compensatory activation of Myt1L and Myt3 transcription in the endocrine compartment, underscoring the quality of gene expression surveillance control. Third, differentiated islet cells maintain Myt1 and Ngn3 expression, which contributes to endocrine maturation and function, indicating that genetic networks that initiate islet cell neogenesis could also participate in endocrine maturation and function. Fourth, Ngn3hi cells were heterogeneous cells that express either high or low levels of Myt1, demonstrating the inherent heterogeneity of the endocrine progenitors. Finally, we have successfully devised a novel bipartite-Cre- based cell lineage tracing method, so that cells that co-express dual protein markers could be specifically labeled. These findings lead to, and provide the tool to test, the hypothesis that the Myt1 interacts with Ngn3 to promote endocrine commitment and islet cell maturation and that differential Myt1 expression divides Ngn3hi progenitors into functionally distinct subtypes. In this grant proposal, we will first determine whether Myt1 is required for high Ngn3 expression and endocrine maturation using loss-of-function studies. Then, we will examine whether Myt1 expression in Ngn3-expressing cells marks them as progenitors of specific islet cell types. These proposed studies are of fundamental significance to cell differentiation in all organs, and also to translational efforts related to pancreas defects/malfunction.

Public Health Relevance

This grant proposal studies how combined gene activities promote endocrine islet cell differentiation, maturation and specification. Results from these studies could lay the foundation to derive functional islet cells for cell-based diabetes therapy. It will also establish a novel technology for cell lineage and cell type specific gene manipulation studies in the field of organogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK065949-10
Application #
8511607
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Sato, Sheryl M
Project Start
2003-12-15
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
10
Fiscal Year
2013
Total Cost
$320,633
Indirect Cost
$114,922
Name
Vanderbilt University Medical Center
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Liu, Jing; Willet, Spencer G; Bankaitis, Eric D et al. (2013) Non-parallel recombination limits Cre-LoxP-based reporters as precise indicators of conditional genetic manipulation. Genesis 51:436-42
Maia, Ana Rita Ramada; Zhu, Xiaodong; Miller, Paul et al. (2013) Modulation of Golgi-associated microtubule nucleation throughout the cell cycle. Cytoskeleton (Hoboken) 70:32-43
Ray, Kevin C; Bell, Kayla M; Yan, Jingbo et al. (2011) Epithelial tissues have varying degrees of susceptibility to Kras(G12D)-initiated tumorigenesis in a mouse model. PLoS One 6:e16786
Wang, Sui; Yan, Jingbo; Anderson, Daniel A et al. (2010) Neurog3 gene dosage regulates allocation of endocrine and exocrine cell fates in the developing mouse pancreas. Dev Biol 339:26-37
Wang, Sui; Jensen, Jan N; Seymour, Philip A et al. (2009) Sustained Neurog3 expression in hormone-expressing islet cells is required for endocrine maturation and function. Proc Natl Acad Sci U S A 106:9715-20
Artner, Isabella; Hang, Yan; Guo, Min et al. (2008) MafA is a dedicated activator of the insulin gene in vivo. J Endocrinol 198:271-9
Wang, Sui; Hecksher-Sorensen, Jacob; Xu, Yanwen et al. (2008) Myt1 and Ngn3 form a feed-forward expression loop to promote endocrine islet cell differentiation. Dev Biol 317:531-40
Means, Anna L; Xu, Yanwen; Zhao, Aizhen et al. (2008) A CK19(CreERT) knockin mouse line allows for conditional DNA recombination in epithelial cells in multiple endodermal organs. Genesis 46:318-23
Xu, Yanwen; Xu, Gang; Liu, Bindong et al. (2007) Cre reconstitution allows for DNA recombination selectively in dual-marker-expressing cells in transgenic mice. Nucleic Acids Res 35:e126
Wang, Sui; Zhang, Jia; Zhao, Aizhen et al. (2007) Loss of Myt1 function partially compromises endocrine islet cell differentiation and pancreatic physiological function in the mouse. Mech Dev 124:898-910

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