SUMMARYCORL proteins (FUSSEL/SKOR proteins in humans) are related to Sno/Ski oncogenes but their developmental roles are unknown. We have cloned Drosophila CORL and show that its expression is restricted to distinct subsets of cells in the central nervous system. We generated a deletion of CORL and noted that homozygous individuals rarely survive to adulthood. Df(4)dCORL adult escapers display mushroom body (MB) defects and Df(4)dCORL larvae are lacking Ecdysone Receptor (EcR-B1) expression in MB neurons. This is phenocopied in CORL-RNAi and Smad2-RNAi clones in wild-type larvae. Furthermore, constitutively active Baboon (type I receptor upstream of Smad2) cannot stimulate EcR-B1 MB expression in Df(4)dCORL larvae, which demonstrates a formal requirement for CORL in Smad2 signaling. Studies of mouse Corl1 (Skor1) revealed that it binds specifically to Smad3. Overall, the data suggest that CORL facilitates Smad2 activity upstream of EcR-B1 in the MB. The conservation of neural expression and strong sequence homology of all CORL proteins suggests that this is a new family of Smad co-factors.

Progenitor cells in the adult pancreas are potential sources of endocrine beta cells for treating type 1 diabetes. Previously, we identified tri-potent progenitor cells in the adult (2–4 month-old) murine pancreas that were capable of self-renewal and differentiation into duct, acinar, and endocrine cells in vitro. These progenitor cells were named pancreatic colony-forming units (PCFUs). However, because PCFUs are a minor population in the pancreas (~1%) they are difficult to study. To enrich PCFUs, strategies using cell-surface marker analyses and fluorescence-activated cell sorting were developed. We found that CD133highCD71low cells, but not other cell populations, enriched PCFUs by up to 30 fold compared to the unsorted cells. CD133highCD71low cells generated primary, secondary, and subsequent colonies when serially re-plated in Matrigel-containing cultures, suggesting self-renewal abilities. In the presence of a laminin hydrogel, CD133highCD71low cells gave rise to colonies that contained duct, acinar, and Insulin+Glucagon+ double-hormonal endocrine cells. Colonies from the laminin hydrogel culture were implanted into diabetic mice, and five weeks later duct, acinar, and Insulin+Glucagon− cells were detected in the grafts, demonstrating tri-lineage differentiation potential of CD133highCD71low cells. These CD133highCD71low cells will enable future studies of putative adult pancreas stem cells in vivo.

SUMMARYThe ability of secreted Transforming Growth Factor  (TGF) proteins to act as morphogens dictates that their influence be strictly regulated. Here, we report that maternally contributed fat facets (faf; a homolog of USP9X/FAM) is essential for proper interpretation of the zygotic Decapentaplegic (Dpp) morphogen gradient that patterns the embryonic dorsal-ventral axis. The data suggest that the loss of faf reduces the activity of Medea (a homolog of Smad4) below the minimum necessary for adequate Dpp signaling and that this is likely due to excessive ubiquitylation on a specific lysine. This study supports the hypothesis that the control of cellular responsiveness to TGF signals at the level of Smad4 ubiquitylation is a conserved mechanism required for proper implementation of a morphogen gradient.

The Sno oncogene (Snoo or dSno in Drosophila) is a highly conserved protein and a well-established antagonist of Transforming Growth Factor-β signaling in overexpression assays. However, analyses of Sno mutants in flies and mice have proven enigmatic in revealing developmental roles for Sno proteins. Thus, to identify developmental roles for dSno we first reconciled conflicting data on the lethality of dSno mutations. Then we conducted analyses of wing development in dSno loss of function genotypes. These studies revealed ectopic margin bristles and ectopic campaniform sensilla in the anterior compartment of the wing blade suggesting that dSno functions to antagonize Wingless (Wg) signaling. A subsequent series of gain of function analyses yielded the opposite phenotype (loss of bristles and sensilla) and further suggested that dSno antagonizes Wg signal transduction in target cells. To date Sno family proteins have not been reported to influence the Wg pathway during development in any species. Overall our data suggest that dSno functions as a tissue-specific component of the Wg signaling pathway with modest antagonistic activity under normal conditions but capable of blocking significant levels of extraneous Wg, a role that may be conserved in vertebrates.

Adult pancreatic stem and progenitor cells may serve as an alternative source of insulin-secreting endocrine cells in cell replacement therapy for type 1 diabetes, but much remained unknown about these cells. We previously identified adult murine pancreatic progenitor-like cells that displayed in vitro self-renewal and trilineage differentiation activities in a three-dimensional colony/organoid assay containing 1% methylcellulose and 5% Matrigel. However, the presence of other undefined culture components, such as serum and conditioned medium, has prevented a complete understanding of the signals required for progenitor cell growth. Here, we have established a serum-free, conditioned medium-free colony assay with the inclusion of seven defined factors: epidermal growth factor (EGF), R-Spondin 1 (RSPO1), Noggin, nicotinamide, exendin-4, activin B, and vascular endothelial growth factor (VEGF)-A. The requirements for colony growth were characterized and we found that EGF and nicotinamide were necessary and sufficient for the colony growth and long-term selfrenewal of these progenitors. However, the seven factor (7F) culture medium better induced colony size and self-renewal in long-term culture than EGF plus nicotinamide alone. Individual 3-week-old colonies grown in the 7F culture medium expressed ductal, acinar, and endocrine lineage markers, suggesting that tri-lineage differentiation of the tri-potent progenitors was occurring without genetic manipulation. A delayed inhibition of Notch signaling using small molecules in 2-week-old cultures enhanced endocrine gene expression in 3-weekold colonies. This better-defined colony assay system will enable our and other laboratories for in-depth mechanistic studies on the biology of these progenitor cells.