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Novel signals guiding endodermal progenitor cells toward a pancreatic fate

Final Report Summary - ENDOPANC (Novel signals guiding endodermal progenitor cells toward a pancreatic fate)

Current and future work in my laboratory aims to elucidate the mechanisms underlying pancreas organogenesis, including fate specification, plasticity and morphogenesis, and to translate these concepts in the understanding of pathogenesis as well as treatment options of diabetes.
The ENDOPANC project has focused on the biological function of the novel pancreatic factor, Shirin/Stard13 in mammalian systems.
• Aim 1: In vivo study of the biological function of Shirin/Stard13 by loss-of-function approach in the mouse.
The development of functional organ architectures relies on coordinated morphogenesis and growth. In the developing pancreas, the branching epithelium is organized in discrete domains, delineating one specific domain of progenitor cells at the tip of the branches. There is currently not much information about how branching occurs and is coordinated with progenitor proliferation in the pancreas. We have started to shed light on these questions, by investigating the role of Rho GTPase and its regulator the RhoGAP protein, Stard13, that we previously identified as an early pancreatic factor. Conditional ablation of Stard13 expression in the mouse developing pancreas disrupts epithelial morphogenesis and tip domain organization, resulting in hampered proliferation of tip progenitor cells and, subsequent, organ hypoplasia. We have shown that Stard13 integrates these events by exerting negative control over Rho signaling during pancreas development. Our findings not only identify a function of the RhoGAP Stard13 in conferring spatio-temporal regulation on the ubiquitous Rho GTPase in the pancreas, but also offer new insights into the mechanisms by which the pancreatic epithelium shapes itself to create a mature organ, linking Rho control of epithelial remodeling to pancreas organ size determination. This original study has been recently published in the journal Development [Petzold K, Naumann H, Spagnoli FM. “Rho signaling restriction by the RhoGAP Stard13 integrates growth and morphogenesis in the pancreas” Development 2013; Vol. 140: 126-135]. In addition, I have been invited to write a short review and commentary on this publication [Zygmunt T, Spagnoli FM. "RhoGAP control of pancreas development. Putting cells in the right place at the right time" Small GTPases 2013; Vol. 19: 4(2)].
To study morphogenesis in my laboratory, we devised an ex vivo mouse pancreatic explant culture method combined with high-resolution imaging that was recently published [Petzold K, Spagnoli FM. “A System for Ex vivo Culturing of Embryonic Pancreas” J. Vis. Exp. 2012; Vol. 27; 66]. This provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing embryo. Future work is aimed at further dissection of the mechanism of action of Stard13 during pancreas morphogenesis.
In parallel, we are studying the metabolic phenotype of adult Stard13 mutant mice, which display glucose intolerance due to disorganized cytosketelon and hampered insulin secretion (Nauman and Spagnoli, unpublished results). Importantly, Stard13 expression in the pancreas becomes restricted to the endocrine islet after birth. Preliminary results in my laboratory have shown that the Stard13 mutant mice display glucose intolerance in the adult age. Future work is aimed at further characterization of the defects in the adult pancreatic tissue and the cause of glucose intolerance.
• Aim 2: In vitro study of Shirin/Stard13 potential activity in triggering differentiation towards pancreatic fate in mouse ES cells.
We established culture conditions for differentiating the mouse ESC model system into endoderm and pancreatic progenitor cells. This represents an excellent an ex-vivo model system for studying cell fate decision and lineage-specific developmental program. In addition, we have generated a conditional gene expression system to induce Shirin/Stard13 expression in mESCs upon differentiation into definitive endoderm. Subsequently, we will examine the differentiation potentials of Stard13+ cells along endodermal lineages, in particular for the induction of pancreatic fate.
In addition, this ex-vivo system is suitable for pursuing structure-function analysis and dissecting the biochemistry of the RhoGAP and START conserved domains of Shirin. To this aim, we have generated two different polyclonal antibodies against Stard13. This represents a crucial step for the advances of our future work on Shirin/Stard13, allowing us to perform co-localization and sub-cellular analysis within the pancreatic tissue and future biochemical analyses aimed at the understanding of the mechanism of action of this novel pancreatic factor. This project is still ongoing in my laboratory. The ex-vivo approaches will provide insights into how and exactly at which step of the pancreatic cell lineage differentiation program Shirin/Stard13 is acting and complement the in vivo analysis of Stard13 conditional knockout mice.
In conclusion, better comprehension of the signals necessary for maintenance of progenitor pool or initiation of differentiation into particular lineage will have practical implications in any attempt of cell-based therapy of diabetes based on expansion of pancreatic progenitors and generation of insulin producing β-cells from stem cells or progenitor cells.

Transfer of knowledge
I am part of the Cardiovascular and Metabolic Research Program at the MDC and have a partnership with the Endocrinology Department at the Charité University, Berlin Medical School Campus. I am faculty member of two Graduate Schools and, recently, have also started to serve as faculty in two Master Programs.
Throughout the academic year, I give lectures for students and postdoctoral fellows at the Helmholtz-Transcard Graduate Program; Helmholtz-Molecular and Cell Biology Graduate Program; “Cell Signaling“ lecture series at the DFG Graduiertenkolleg 1772 Computational Systems Biology; and DFG-Molecular Endocrinology Graduate program of the Charité-Universitätsmedizin.