We will generate translationally essential information on the process of ?-cell differentiation from progenitor cells, targeting a major outstanding issue: what stimuli and culture conditions to employ to produce mature ?-cells in vitro. Our team will focus solely on the tissue source of all ?-cell progenitors: the trunk domain epithelium of mid-pancreas organogenesis. The spatiotemporally orchestrated signaling networks and cellular division/migration processes that control ?-cell birth/maturation are very poorly defined. But, cell-autonomous and non-autonomous programs that give rise to mature b cells must work within the constraints of a defined epithelial structure, with its tightly linked morphogenetic program that produces the large numbers of budded, mature islets. We will perform a high-resolution structural and cell biological analysis of endocrine progenitor locations, cell-cell organization and signaling, and their behavior in yielding differentiating ?-cell progeny or remaining as cycling endocrine-biased cells within the epithelium. We will: (1) Define how the epithelial structure provides signaling niches that control the location, number and behavior of endocrine-biased progenitors. (2) Define how asymmetric division yields pro-endocrine precursors and endocrine-committed progeny. (3) Characterize the directional delamination of pro-endocrine cells and their progression to ?-cell or other endocrine types. (4) Localize intermediates marked by cell-instructive factors, and determine cell-autonomous and cell-non-autonomous effects. (5) Use mutational analysis and lineage tracing to detail the intraepithelial organization and signaling processes involved. (6) Perform gene expression profiling of selected cell populations in normal and perturbed situations to provide an unprecedented understanding of the gene regulatory networks involved, plus new insights into markers of critical transitional cell states. (7) Map new monoclonal antibodies from the BCBC cell-surface-marker project onto this framework, providing new tools for identifying/sorting progenitor-progeny states in differentiating hESC. (8) Test directly the functional information emerging from our studies as ?-cell formation/maturation stimuli on human ES cell (hESC) differentiating in vitro. Such a spatiotemporally resolved 'normal differentiation framework'will shed light on deficiencies in hESC differentiation protocols. In vitro hESC-derived ?-cells are immature and low in number, while partly differentiated, propancreatic cell clusters produce functional ?-cells on maturation in mice: we hypothesize that the in vitro deficit reflects an inability to build proper communication and progenitor qualities. We designed our project to integrate with and complement studies of other BCBC teams on hESC differentiation and endocrine progenitor identification. The team's pedigree in lineage analysis, cell biology and progenitor control, longevity and strong intra-BCBC interactions, led to our sharply defined common interest in epithelial endocrine progenitors. Novel tools, reagents, data sets will be shared with all BCBC members.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01DK089570-03
Application #
8316317
Study Section
Special Emphasis Panel (ZDK1-GRB-G (M3))
Program Officer
Sato, Sheryl M
Project Start
2010-09-15
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
3
Fiscal Year
2012
Total Cost
$1,378,869
Indirect Cost
$253,249
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
Greggio, Chiara; De Franceschi, Filippo; Grapin-Botton, Anne (2015) Concise reviews: In vitro-produced pancreas organogenesis models in three dimensions: self-organization from few stem cells or progenitors. Stem Cells 33:14-Aug
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Willet, Spencer G; Hale, Michael A; Grapin-Botton, Anne et al. (2014) Dominant and context-specific control of endodermal organ allocation by Ptf1a. Development 141:4385-94
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Baeyens, Luc; Lemper, Marie; Leuckx, Gunter et al. (2014) Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice. Nat Biotechnol 32:76-83
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von Figura, Guido; Morris 4th, John P; Wright, Christopher V E et al. (2014) Nr5a2 maintains acinar cell differentiation and constrains oncogenic Kras-mediated pancreatic neoplastic initiation. Gut 63:656-64
Greggio, Chiara; De Franceschi, Filippo; Figueiredo-Larsen, Manuel et al. (2013) Artificial three-dimensional niches deconstruct pancreas development in vitro. Development 140:4452-62
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
Meredith, David M; Borromeo, Mark D; Deering, Tye G et al. (2013) Program specificity for Ptf1a in pancreas versus neural tube development correlates with distinct collaborating cofactors and chromatin accessibility. Mol Cell Biol 33:3166-79

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