SUMMARYOne major unresolved question in the field of pancreas biology is whether ductal cells have the ability to generate insulinproducing b-cells. Conclusive examination of this question has been limited by the lack of appropriate tools to efficiently and specifically label ductal cells in vivo. We generated Sox9CreER T2 mice, which, during adulthood, allow for labeling of an average of 70% of pancreatic ductal cells, including terminal duct/centroacinar cells. Fate-mapping studies of the Sox9 + domain revealed endocrine and acinar cell neogenesis from Sox9 + cells throughout embryogenesis. Very small numbers of non-b endocrine cells continue to arise from Sox9 + cells in early postnatal life, but no endocrine or acinar cell neogenesis from Sox9 + cells occurs during adulthood. In the adult pancreas, pancreatic injury by partial duct ligation (PDL) has been suggested to induce b-cell regeneration from a transient Ngn3 + endocrine progenitor cell population. Here, we identify ductal cells as a cell of origin for PDL-induced Ngn3 + cells, but fail to observe b-cell neogenesis from duct-derived cells. Therefore, although PDL leads to activation of Ngn3 expression in ducts, PDL does not induce appropriate cues to allow for completion of the entire b-cell neogenesis program. In conclusion, although endocrine cells arise from the Sox9 + ductal domain throughout embryogenesis and the early postnatal period, Sox9 + ductal cells of the adult pancreas no longer give rise to endocrine cells under both normal conditions and in response to PDL.

Lung branching morphogenesis is a highly orchestrated process that gives rise to the complex network of gas-exchanging units in the adult lung. Intricate regulation of signaling pathways, transcription factors, and epithelial-mesenchymal cross-talk are critical to ensuring branching morphogenesis occurs properly. Here, we describe a role for the transcription factor Sox9 during lung branching morphogenesis. Sox9 is expressed at the distal tips of the branching epithelium in a highly dynamic manner as branching occurs and is down-regulated starting at embryonic day 16.5, concurrent with the onset of terminal differentiation of type 1 and type 2 alveolar cells. Using epithelial-specific genetic loss-and gain-of-function approaches, our results demonstrate that Sox9 controls multiple aspects of lung branching. Fine regulation of Sox9 levels is required to balance proliferation and differentiation of epithelial tip progenitor cells, and loss of Sox9 leads to direct and indirect cellular defects including extracellular matrix defects, cytoskeletal disorganization, and aberrant epithelial movement. Our evidence shows that unlike other endoderm-derived epithelial tissues, such as the intestine, Wnt/β-catenin signaling does not regulate Sox9 expression in the lung. We conclude that Sox9 collectively promotes proper branching morphogenesis by controlling the balance between proliferation and differentiation and regulating the extracellular matrix.organogenesis | campomelic dysplasia

In the pancreas, Notch signaling is thought to prevent cell differentiation, thereby maintaining progenitors in an undifferentiated state. Here, we show that Notch renders progenitors competent to differentiate into ductal and endocrine cells by inducing activators of cell differentiation. Notch signaling promotes the expression of Sox9, which cell-autonomously activates the pro-endocrine gene Ngn3. However, at high Notch activity endocrine differentiation is blocked, as Notch also induces expression of the Ngn3 repressor Hes1. At the transition from high to intermediate Notch activity, only Sox9, but not Hes1, is maintained, thus de-repressing Ngn3 and initiating endocrine differentiation. In the absence of Sox9 activity, endocrine and ductal cells fail to differentiate, resulting in polycystic ducts devoid of primary cilia. Although Sox9 is required for Ngn3 induction, endocrine differentiation necessitates subsequent Sox9 downregulation and evasion from Notch activity via cell-autonomous repression of Sox9 by Ngn3. If high Notch levels are maintained, endocrine progenitors retain Sox9 and undergo ductal fate conversion. Taken together, our findings establish a novel role for Notch in initiating both ductal and endocrine development and reveal that Notch does not function in an on-off mode, but that a gradient of Notch activity produces distinct cellular states during pancreas development.

The pancreas is an essential organ for proper nutrient metabolism and has both endocrine and exocrine function. In the past two decades, knowledge of how the pancreas develops during embryogenesis has significantly increased, largely from developmental studies in model organisms. Specifically, the molecular basis of pancreatic lineage decisions and cell differentiation is well studied. Still not well understood are the mechanisms governing three-dimensional morphogenesis of the organ. Strategies to derive transplantable β-cells in vitro for diabetes treatment have benefited from the accumulated knowledge of pancreas development. In this review, we provide an overview of the current understanding of pancreatic lineage determination and organogenesis, and we examine future implications of these findings for treatment of diabetes mellitus through cell replacement.

The study of hematopoietic colony-forming units using semisolid culture media has greatly advanced the knowledge of hematopoiesis. Here we report that similar methods can be used to study pancreatic colony-forming units. We have developed two pancreatic colony assays that enable quantitative and functional analyses of progenitor-like cells isolated from dissociated adult (2-4 mo old) murine pancreas. We find that a methylcellulose-based semisolid medium containing Matrigel allows growth of duct-like "Ring/ Dense" colonies from a rare (∼1%) population of total pancreatic single cells. With the addition of roof plate-specific spondin 1, a wingless-int agonist, Ring/Dense colony-forming cells can be expanded more than 100,000-fold when serially dissociated and replated in the presence of Matrigel. When cells grown in Matrigel are then transferred to a Matrigel-free semisolid medium with a unique laminin-based hydrogel, some cells grow and differentiate into another type of colony, which we name "Endocrine/Acinar." These Endocrine/Acinar colonies are comprised mostly of endocrine-and acinar-like cells, as ascertained by RNA expression analysis, immunohistochemistry, and electron microscopy. Most Endocrine/Acinar colonies contain beta-like cells that secrete insulin/C-peptide in response to D-glucose and theophylline. These results demonstrate robust self-renewal and differentiation of adult Ring/Dense colony-forming units in vitro and suggest an approach to producing beta-like cells for cell replacement of type 1 diabetes. The methods described, which include microfluidic expression analysis of single cells and colonies, should also advance study of pancreas development and pancreatic progenitor cells. extracellular matrix proteins | Sry-related HMG box (Sox) 9 | Promonin 1 (CD133) | neurogenin 3 | dickkopf1 (Dkk1)

Pitx2 expression is observed during all states of the myogenic progression in embryonic muscle anlagen and persists in adult muscle. Pitx2 mutant mice form all but a few muscle anlagen. Loss or degeneration in muscle anlagen could generally be attributed to the loss of a muscle attachment site induced by some other aspect of the Pitx2 phenotype. Muscles derived from the first branchial arch were absent, whereas muscles derived from the second branchial arch were merely distorted in Pitx2 mutants at midgestation. Pitx2 was expressed well before, and was required for, initiation of the myogenic progression in the first, but not second, branchial arch mesoderm. Pitx2 was also required for expression of premyoblast specification markers Tbx1, Tcf21, and Msc in the first, but not second, branchial arch. First, but not second, arch mesoderm of Pitx2 mutants failed to enlarge after embryonic day 9.5, well before the onset of the myogenic progression. Thus, Pitx2 contributes to specification of first, but not second, arch mesoderm. The jaw of Pitx2 mutants was vestigial by midgestation, but significant size reductions were observed as early as embryonic day 10.5. The diminutive first branchial arch of mutants could not be explained by loss of mesoderm alone, suggesting that Pitx2 contributes to the earliest specification of jaw itself.homeobox gene ͉ muscle development

SUMMARYAll mature pancreatic cell types arise from organ-specific multipotent progenitor cells. Although previous studies have identified cell-intrinsic and -extrinsic cues for progenitor cell expansion, it is unclear how these cues are integrated within the niche of the developing organ. Here, we present genetic evidence in mice that the transcription factor Sox9 forms the centerpiece of a gene regulatory network that is crucial for proper organ growth and maintenance of organ identity. We show that pancreatic progenitorspecific ablation of Sox9 during early pancreas development causes pancreas-to-liver cell fate conversion. Sox9 deficiency results in cell-autonomous loss of the fibroblast growth factor receptor (Fgfr) 2b, which is required for transducing mesenchymal Fgf10 signals. Likewise, Fgf10 is required to maintain expression of Sox9 and Fgfr2 in epithelial progenitors, showing that Sox9, Fgfr2 and Fgf10 form a feed-forward expression loop in the early pancreatic organ niche. Mirroring Sox9 deficiency, perturbation of Fgfr signaling in pancreatic explants or genetic inactivation of Fgf10 also result in hepatic cell fate conversion. Combined with previous findings that Fgfr2b or Fgf10 are necessary for pancreatic progenitor cell proliferation, our results demonstrate that organ fate commitment and progenitor cell expansion are coordinately controlled by the activity of a Sox9/Fgf10/Fgfr2b feed-forward loop in the pancreatic niche. This self-promoting Sox9/Fgf10/Fgfr2b loop may regulate cell identity and organ size in a broad spectrum of developmental and regenerative contexts.

Summary During pancreas development, epithelial buds undergo branching morphogenesis to form an exocrine and endocrine gland. Proper morphogenesis is necessary for correct lineage allocation of pancreatic progenitors; however, the cellular events underlying pancreas morphogenesis are unknown. Here, we employed time-lapse microscopy and fluorescent labeling of cells to analyze cell behaviors associated with pancreas morphogenesis. We observed that outer bud cells adjacent to the basement membrane are pleomorphic and rearrange frequently; as well, they largely remain in the outer cell compartment even after mitosis. These cell behaviors and pancreas branching depend on cell contacts with the basement membrane, which induce actomyosin cytoskeleton remodeling via integrin-mediated activation of FAK/Src signaling. We show that integrin signaling reduces E-cadherin-mediated cell-cell adhesion in outer cells, and provide genetic evidence that this regulation is necessary for initiation of branching. Our study suggests that regulation of cell motility and adhesion by local niche cues initiates pancreas branching morphogenesis.