Maike Sander

SOX9 is required for maintenance of the pancreatic progenitor cell pool

The factors necessary to maintain organ-specific progenitor cells are poorly understood and yet of extreme clinical importance. Here, we identify the transcription factor SOX9 as the first specific marker and maintenance factor of multipotential progenitors during pancreas organogenesis. In the developing pancreas, SOX9 expression is restricted to a mitotically active, Notch-responsive subset of PDX1+ pluripotent progenitors and is absent from committed endocrine precursors or differentiated cells. Similar to Notch mutations, organ-specific Sox9 inactivation in mice causes severe pancreatic hypoplasia resulting from depletion of the progenitor cell pool. We show that Sox9 maintains pancreatic progenitors by stimulating their proliferation, survival, and persistence in an undifferentiated state. Our finding that SOX9 regulates the Notch-effector HES1 suggests a Notch-dependent mechanism and establishes a possible genetic link between SOX factors and Notch. These findings will be of major significance for the development of in vitro protocols for cell replacement therapies.

Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas.

One major unresolved question in the field of pancreas biology is whether ductal cells have the ability to generate insulin-producing β-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 Sox9CreERT2 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-β 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 β-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 β-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 β-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.

Identification of Sox9-Dependent Acinar-to-Ductal Reprogramming as the Principal Mechanism for Initiation of Pancreatic Ductal Adenocarcinoma

Tumors are largely classified by histologic appearance, yet morphologic features do not necessarily predict cellular origin. To determine the origin of pancreatic ductal adenocarcinoma (PDA), we labeled and traced pancreatic cell populations after induction of a PDA-initiating Kras mutation. Our studies reveal that ductal and stem-like centroacinar cells are surprisingly refractory to oncogenic transformation, whereas acinar cells readily form PDA precursor lesions with ductal features. We show that formation of acinar-derived premalignant lesions depends on ectopic induction of the ductal gene Sox9. Moreover, when concomitantly expressed with oncogenic Kras, Sox9 accelerates formation of premalignant lesions. These results provide insight into the cellular origin of PDA and suggest that its precursors arise via induction of a duct-like state in acinar cells.

Generation of oligodendrocyte precursor cells from mouse dorsal spinal cord independent of Nkx6 regulation and Shh signaling.

In the developing spinal cord, early progenitor cells of the oligodendrocyte lineage are induced in the motor neuron progenitor (pMN) domain of the ventral neuroepithelium by the ventral midline signal Sonic hedgehog (Shh). The ventral generation of oligodendrocytes requires Nkx6-regulated expression of the bHLH gene Olig2 in this domain. In the absence of Nkx6 genes or Shh signaling, the initial expression of Olig2 in the pMN domain is completely abolished. In this study, we provide the in vivo evidence for a late phase of Olig gene expression independent of Nkx6 and Shh gene activities and reveal a brief second wave of oligodendrogenesis in the dorsal spinal cord. In addition, we provide genetic evidence that oligodendrogenesis can occur in the absence of hedgehog receptor Smoothened, which is essential for all hedgehog signaling.

Human β Cell Transcriptome Analysis Uncovers lncRNAs That Are Tissue-Specific, Dynamically Regulated, and Abnormally Expressed in Type 2 Diabetes

A significant portion of the genome is transcribed as long noncoding RNAs (lncRNAs), several of which are known to control gene expression. The repertoire and regulation of lncRNAs in disease-relevant tissues, however, has not been systematically explored. We report a comprehensive strand-specific transcriptome map of human pancreatic islets and β cells, and uncover >1100 intergenic and antisense islet-cell lncRNA genes. We find islet lncRNAs that are dynamically regulated and show that they are an integral component of the β cell differentiation and maturation program. We sequenced the mouse islet transcriptome and identify lncRNA orthologs that are regulated like their human counterparts. Depletion of HI-LNC25, a β cell-specific lncRNA, downregulated GLIS3 mRNA, thus exemplifying a gene regulatory function of islet lncRNAs. Finally, selected islet lncRNAs were dysregulated in type 2 diabetes or mapped to genetic loci underlying diabetes susceptibility. These findings reveal a new class of islet-cell genes relevant to β cell programming and diabetes pathophysiology.

Inactivation of specific β cell transcription factors in type 2 diabetes

Type 2 diabetes (T2DM) commonly arises from islet β cell failure and insulin resistance. Here, we examined the sensitivity of key islet-enriched transcription factors to oxidative stress, a condition associated with β cell dysfunction in both type 1 diabetes (T1DM) and T2DM. Hydrogen peroxide treatment of β cell lines induced cytoplasmic translocation of MAFA and NKX6.1. In parallel, the ability of nuclear PDX1 to bind endogenous target gene promoters was also dramatically reduced, whereas the activity of other key β cell transcriptional regulators was unaffected. MAFA levels were reduced, followed by a reduction in NKX6.1 upon development of hyperglycemia in db/db mice, a T2DM model. Transgenic expression of the glutathione peroxidase-1 antioxidant enzyme (GPX1) in db/db islet β cells restored nuclear MAFA, nuclear NKX6.1, and β cell function in vivo. Notably, the selective decrease in MAFA, NKX6.1, and PDX1 expression was found in human T2DM islets. MAFB, a MAFA-related transcription factor expressed in human β cells, was also severely compromised. We propose that MAFA, MAFB, NKX6.1, and PDX1 activity provides a gauge of islet β cell function, with loss of MAFA (and/or MAFB) representing an early indicator of β cell inactivity and the subsequent deficit of more impactful NKX6.1 (and/or PDX1) resulting in overt dysfunction associated with T2DM.

Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice

The molecular identification of adult hepatic stem/progenitor cells has been hampered by the lack of truly specific markers. To isolate putative adult liver progenitor cells, we used cell surface-marking antibodies, including MIC1-1C3, to isolate subpopulations of liver cells from normal adult mice or those undergoing an oval cell response and tested their capacity to form bilineage colonies in vitro. Robust clonogenic activity was found to be restricted to a subset of biliary duct cells antigenically defined as CD45(-)/CD11b(-)/CD31(-)/MIC1-1C3(+)/CD133(+)/CD26(-), at a frequency of one of 34 or one of 25 in normal or oval cell injury livers, respectively. Gene expression analyses revealed that Sox9 was expressed exclusively in this subpopulation of normal liver cells and was highly enriched relative to other cell fractions in injured livers. In vivo lineage tracing using Sox9creER(T2)-R26R(YFP) mice revealed that the cells that proliferate during progenitor-driven liver regeneration are progeny of Sox9-expressing precursors. A comprehensive array-based comparison of gene expression in progenitor-enriched and progenitor-depleted cells from both normal and DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine or diethyl1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate)-treated livers revealed new potential regulators of liver progenitors.

Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells.

UNLABELLED BACKGROUND& AIMS: Embryonic biliary precursor cells form a periportal sheet called the ductal plate, which is progressively remodeled to generate intrahepatic bile ducts. A limited number of ductal plate cells participate in duct formation; those not involved in duct development are believed to involute by apoptosis. Moreover, cells that express the SRY-related HMG box transcription factor 9 (SOX9), which include the embryonic ductal plate cells, were proposed to continuously supply the liver with hepatic cells. We investigated the role of the ductal plate in hepatic morphogenesis. METHODS Apoptosis and proliferation were investigated by immunostaining of mouse and human fetal liver tissue. The postnatal progeny of SOX9-expressing ductal plate cells was analyzed after genetic labeling, at the ductal plate stage, by Cre-mediated recombination of a ROSA26RYFP reporter allele. Inducible Cre expression was induced by SOX9 regulatory regions, inserted in a bacterial artificial chromosome. Livers were studied from mice under normal conditions and during diet-induced regeneration. RESULTS Ductal plate cells did not undergo apoptosis and showed limited proliferation. They generated cholangiocytes lining interlobular bile ducts, bile ductules, and canals of Hering, as well as periportal hepatocytes. Oval cells that appeared during regeneration also derived from the ductal plate. We did not find that liver homeostasis required a continuous supply of cells from SOX9-expressing progenitors. CONCLUSIONS The ductal plate gives rise to cholangiocytes lining the intrahepatic bile ducts, including its most proximal segments. It also generates periportal hepatocytes and adult hepatic progenitor cells.

Nkx6 Transcription Factors and Ptf1a Function as Antagonistic Lineage Determinants in Multipotent Pancreatic Progenitors

The molecular mechanisms that underlie cell lineage diversification of multipotent progenitors in the pancreas are virtually unknown. Here we show that the early fate choice of pancreatic progenitors between the endocrine and acinar cell lineage is restricted by cross-repressive interactions between the transcription factors Nkx6.1/Nkx6.2 (Nkx6) and Ptf1a. Using genetic loss- and gain-of-function approaches, we demonstrate that Nkx6 factors and Ptf1a are required and sufficient to repress the alternative lineage program and to specify progenitors toward an endocrine or acinar fate, respectively. The Nkx6/Ptf1a switch only operates during a critical competence window when progenitors are still multipotent and can be uncoupled from cell differentiation. Thus, cross-antagonism between Nkx6 and Ptf1a in multipotent progenitors governs the equilibrium between endocrine and acinar cell neogenesis required for normal pancreas development.

NKX6 transcription factor activity is required for α- andβ -cell development in the pancreas

In diabetic individuals, the imbalance in glucose homeostasis is caused by loss or dysfunction of insulin-secreting β-cells of the pancreatic islets. As successful generation of insulin-producing cells in vitro could constitute a cure for diabetes, recent studies have explored the molecular program that underlies β-cell formation. From these studies, the homeodomain transcription factor NKX6.1 has proven to be a key player. In Nkx6.1 mutants, β-cell numbers are selectively reduced, while other islet cell types develop normally. However, the molecular events downstream of NKX6.1, as well as the molecular pathways that ensure residualβ -cell formation in the absence of NKX6.1 are largely unknown. Here, we show that the Nkx6.1 paralog, Nkx6.2, is expressed during pancreas development and partially compensates for NKX6.1 function. Surprisingly, our analysis of Nkx6 compound mutant mice revealed a previously unrecognized requirement for NKX6 activity in α-cell formation. This finding suggests a more general role for NKX6 factors in endocrine cell differentiation than formerly suggested. Similar to NKX6 factors, the transcription factor MYT1 has recently been shown to regulateα - as well as β-cell development. We demonstrate that expression of Myt1 depends on overall Nkx6 gene dose, and therefore identify Myt1 as a possible downstream target of Nkx6 genes in the endocrine differentiation pathway.