Dicer Regulates Differentiation and Viability during Mouse Pancreatic Cancer Initiation
2014; 9 (5)
Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells
miRNA levels are altered in pancreatic ductal adenocarcinoma (PDA), the most common and lethal pancreatic malignancy, and intact miRNA processing is essential for lineage specification during pancreatic development. However, the role of miRNA processing in PDA has not been explored. Here we study the role of miRNA biogenesis in PDA development by deleting the miRNA processing enzyme Dicer in a PDA mouse model driven by oncogenic Kras. We find that loss of Dicer accelerates Kras driven acinar dedifferentiation and acinar to ductal metaplasia (ADM), a process that has been shown to precede and promote the specification of PDA precursors. However, unconstrained ADM also displays high levels of apoptosis. Dicer loss does not accelerate development of Kras driven PDA precursors or PDA, but surprisingly, we observe that mouse PDA can develop without Dicer, although at the expense of proliferative capacity. Our data suggest that intact miRNA processing is involved in both constraining pro-tumorigenic changes in pancreatic differentiation as well as maintaining viability during PDA initiation.
View details for DOI 10.1371/journal.pone.0095486
View details for Web of Science ID 000335510600039
View details for PubMedID 24788257
Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (31): 12691-12696
Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001.
View details for DOI 10.7554/eLife.00940
View details for Web of Science ID 000328641800001
View details for PubMedID 24252877
Genetic interplays between Msx2 and Foxn1 are required for Notch1 expression and hair shaft differentiation
2009; 326 (2): 420-430
Developmental biology is challenged to reveal the function of numerous candidate genes implicated by recent genome-scale studies as regulators of organ development and diseases. Recapitulating organogenesis from purified progenitor cells that can be genetically manipulated would provide powerful opportunities to dissect such gene functions. Here we describe systems for reconstructing pancreas development, including islet β-cell and α-cell differentiation, from single fetal progenitor cells. A strict requirement for native genetic regulators of in vivo pancreas development, such as Ngn3, Arx, and Pax4, revealed the authenticity of differentiation programs in vitro. Efficient genetic screens permitted by this system revealed that Prdm16 is required for pancreatic islet development in vivo. Discovering the function of genes regulating pancreas development with our system should enrich strategies for regenerating islets for treating diabetes mellitus.
View details for DOI 10.1073/pnas.1304507110
View details for Web of Science ID 000322441500050
View details for PubMedID 23852729
Notch-deficient skin induces a lethal systemic B-lymphoproliferative disorder by secreting TSLP, a sentinel for epidermal integrity
2008; 6 (5): 992-1005
Hair shafts are produced from stem cells located in the bulge. Our knowledge of the genetic pathways regulating cell fate acquisition in the immediate descendents of these stem cells, and fate maintenance in their committed progeny, is still incomplete. One pathway involved in fate maintenance within the hair matrix is the Notch pathway. Here we use compound genetic mutants to demonstrate that two transcription factors, Msx2 and Foxn1, are both required to maintain Notch1 expression in the hair follicle matrix. In their absence, Notch1 is markedly reduced in hair matrix; as a consequence, medulla and inner root sheath (IRS) differentiation is impaired. Our studies also suggest that Foxn1 is a direct activator of the Notch1 promoter activity through one or more putative Foxn1 consensus binding sites located within the 4.7 kb of mouse Notch1 promoter. Since recombinant human BMP4 can induce Foxn1 expression in Msx2-deficient hair follicles, and that their effect on cortical keratin expression appears synergistic, we suggest that these two genes function in parallel pathways downstream of BMP signaling and upstream of Notch1. Independent from their role in Notch activation, Msx2 and Foxn1 also contribute to the expression of several cortical and cuticle keratins. The impact of these additional defects is the complete loss of all visible external hairs, not seen in Notch1 mutants. Our results position Msx2 and Foxn1 upstream of Notch1 within the hair matrix and demonstrate that together these factors play a pivotal role in IRS, cortex and medulla differentiation.
View details for DOI 10.1016/j.ydbio.2008.11.021
View details for Web of Science ID 000263285200014
View details for PubMedID 19103190
Murine vibrissae cultured in serum-free medium reinitiate anagen
JOURNAL OF INVESTIGATIVE DERMATOLOGY
2008; 128 (2): 482-485
Bi-compartmental communication contributes to the opposite proliferative behavior of Notch1-deficient hair follicle and epidermal keratinocytes
2007; 134 (15): 2795-2806
Epidermal keratinocytes form a highly organized stratified epithelium and sustain a competent barrier function together with dermal and hematopoietic cells. The Notch signaling pathway is a critical regulator of epidermal integrity. Here, we show that keratinocyte-specific deletion of total Notch signaling triggered a severe systemic B-lymphoproliferative disorder, causing death. RBP-j is the DNA binding partner of Notch, but both RBP-j-dependent and independent Notch signaling were necessary for proper epidermal differentiation and lipid deposition. Loss of both pathways caused a persistent defect in skin differentiation/barrier formation. In response, high levels of thymic stromal lymphopoietin (TSLP) were released into systemic circulation by Notch-deficient keratinocytes that failed to differentiate, starting in utero. Exposure to high TSLP levels during neonatal hematopoiesis resulted in drastic expansion of peripheral pre- and immature B-lymphocytes, causing B-lymphoproliferative disorder associated with major organ infiltration and subsequent death, a previously unappreciated systemic effect of TSLP. These observations demonstrate that local skin perturbations can drive a lethal systemic disease and have important implications for a wide range of humoral and autoimmune diseases with skin manifestations.
View details for DOI 10.1371/journal.pbio.0060123
View details for Web of Science ID 000256850100011
View details for PubMedID 18507503
Organization of the human PTK7 gene encoding a receptor protein tyrosine kinase-like molecule and alternative splicing of its mRNA
BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION
2002; 1579 (2-3): 153-163
Notch1-deficient epidermal keratinocytes become progressively hyperplastic and eventually produce tumors. By contrast, Notch1-deficient hair matrix keratinocytes have lower mitotic rates, resulting in smaller follicles with fewer cells. In addition, the ratio of melanocytes to keratinocytes is greatly reduced in hair follicles. Investigation into the underlying mechanism for these phenotypes revealed significant changes in the Kit, Tgfbeta and insulin-like growth factor (IGF) signaling pathways, which have not been previously shown to be downstream of Notch signaling. The level of Kitl (Scf) mRNA produced by Notch1-deficient follicular keratinocytes was reduced when compared with wild type, resulting in a decline in melanocyte population. Tgfbeta ligands were elevated in Notch1-deficient keratinocytes, which correlated with elevated expression of several targets, including the diffusible IGF antagonist Igfbp3 in the dermal papilla. Diffusible stromal targets remained elevated in the absence of epithelial Tgfbeta receptors, consistent with paracrine Tgfbeta signaling. Overexpression of Igf1 in the keratinocyte reversed the phenotype, as expected if Notch1 loss altered the IGF/insulin-like growth factor binding protein (IGFBP) balance. Conversely, epidermal keratinocytes contained less stromal Igfbp4 and might thus be primed to experience an increase in IGF signaling as animals age. These results suggest that Notch1 participates in a bi-compartmental signaling network that controls homeostasis, follicular proliferation rates and melanocyte population within the skin.
View details for DOI 10.1242/dev.02868
View details for Web of Science ID 000248381600009
View details for PubMedID 17611229
Genetic mosaic analysis indicates that the bulb region of coat hair follicles contains a resident population of several active multipotent epithelial lineage progenitors
2002; 242 (1): 44-57
Protein tyrosine kinase-7 (PTK7) is a receptor protein tyrosine kinase (RPTK)-like molecule that contains a catalytically inactive tyrosine kinase domain. We report here the genomic structure of the human PTK7 gene by screening a BAC library and DNA sequencing. The PTK7 gene is organized into 20 exons. All of the splicing junctions followed the conserved GT/AG rule. The exon-intron structure of the PTK7 gene in the region that encodes the catalytic domain was distinct from those of other RPTKs with strong homology. The 5'-flanking sequence of the PTK7 gene contains two GC boxes that concatenate Sp1 binding motifs, but does not contain either the TATA or CAAT consensus sequence. Using a luciferase reporter assay, it was demonstrated that the 883-bp 5'-flanking sequence is functional as a promoter of the PTK7 gene. We identified four new splicing variants in testis that could be derived from alternative splicing of exons 8-10, 10, a part of 12-13, and 16. The expression patterns of the splicing variants in the hepatoma and colon cancer cells were different from those of the testis. Our findings suggest that PTK7 is evolutionarily distinct from other RPTKs, and that the alternative splicing of PTK7 mRNA may contribute to its diverse function in cell signaling.
View details for Web of Science ID 000179283800009
View details for PubMedID 12427550
Integration of cytogenetic landmarks into the draft sequence of the human genome
2001; 409 (6822): 953-958
The hair follicle represents an excellent model system for exploring the properties of lineage-forming units in a dynamic epithelium containing multiple cell types. During its growth (anagen) phase, the proximal-distal axis of the mouse coat hair (pelage) follicle provides a historical record of all epithelial lineages generated from its resident stem cell population. An unresolved question in the field is whether the bulb region of anagen pelage follicles contains multipotential progenitors and whether their individual contribution to cellular census fluctuates over time. To address this issue, chimeric follicles were harvested in midanagen from three types of genetic mosaic mouse models. Analysis of the distribution of genotypic markers, including digital three-dimensional reconstruction of serially sectioned chimeric follicles, revealed that on average the bulb contains four or fewer active progenitors, each capable of giving rise to all six follicular epithelial fates. Moreover, analysis of mosaic pelage, as well as cultured whisker follicles provided evidence that bulb-associated progenitors can give rise to expanding descendant clones during midanagen, leading to the conclusion that the bulb contains dormant or symmetrically dividing stem cells. This latter feature resembles the behavior of hematopoietic stem cells after bone marrow transplantation, and raises the question of whether this property may be shared by stem cells in other self-renewing epithelia.
View details for DOI 10.1006/dbio.2001.0516
View details for Web of Science ID 000173608100004
View details for PubMedID 11795939
A 12-Mb complete coverage BAC contig map in human chromosome 16p13.1-p11.2
1999; 9 (8): 763-774
We have placed 7,600 cytogenetically defined landmarks on the draft sequence of the human genome to help with the characterization of genes altered by gross chromosomal aberrations that cause human disease. The landmarks are large-insert clones mapped to chromosome bands by fluorescence in situ hybridization. Each clone contains a sequence tag that is positioned on the genomic sequence. This genome-wide set of sequence-anchored clones allows structural and functional analyses of the genome. This resource represents the first comprehensive integration of cytogenetic, radiation hybrid, linkage and sequence maps of the human genome; provides an independent validation of the sequence map and framework for contig order and orientation; surveys the genome for large-scale duplications, which are likely to require special attention during sequence assembly; and allows a stringent assessment of sequence differences between the dark and light bands of chromosomes. It also provides insight into large-scale chromatin structure and the evolution of chromosomes and gene families and will accelerate our understanding of the molecular bases of human disease and cancer.
View details for Web of Science ID 000166938800068
View details for PubMedID 11237021
We have constructed a complete coverage BAC contig map that spans a 12-Mb genomic segment in the human chromosome 16p13.1-p11.2 region. The map consists of 68 previously mapped STSs and 289 BAC clones, 51 of which-corresponding to a total of 7.721 Mb of genomic DNA-have been sequenced, and provides a high resolution physical map of the region. Contigs were initially built based mainly on the analysis of STS contents and restriction fingerprint patterns of the clones. To close the gaps, probes derived from BAC clone ends were used to screen deeper BAC libraries. Clone end sequence data obtained from chromosome 16-specific BACs, as well as from public databases, were used for the identification of BACs that overlap with fully sequenced BACs by means of sequence match. This approach allowed precise alignment of clone overlaps in addition to restriction fingerprint comparison. A freehand contig drawing software tool was developed and used to manage the map data graphically and generate a real scale physical map. The map we present here is approximately 3.5 x deep and provides a minimal tiling path that covers the region in an array of contigous, overlapping BACs.
View details for Web of Science ID 000082063000010
View details for PubMedID 10447511