Impact of Sox9 Dosage and Hes1-mediated Notch Signaling in Controlling the Plasticity of Adult Pancreatic Duct Cells in Mice

Similar documents
Mice( Dissertation_ 全文 ) /doctor.k19

SUPPLEMENTARY DATA. Supplementary Table 2. Antibodies used for Immunofluoresence. Supplementary Table 3. Real-time PCR primer sequences.

Supplementary Figure 1: Hsp60 / IEC mice are embryonically lethal (A) Light microscopic pictures show mouse embryos at developmental stage E12.

Supplementary Figure 1. Satellite cell contribution to myofibers in whole. gastrocnemius/plantaris/soleus, diaphragm, and EOM of 12 or 20 month

Supplementary Table 1. Antibodies and dilutions used in the immunohistochemical study

marker. DAPI labels nuclei. Flies were 20 days old. Scale bar is 5 µm. Ctrl is

Fig. S1. Generation of pancreas-specific Stard13 PA-deleted mice. (A) Schematic representation of the targeted Stard13 flox allele, indicating the


Supplementary Figure 1.

Analysis of the plasticity of duct cells in the embryonic and adult pancreas. Myriam Solar Hospital Clinic de Barcelona Barcelona 2009

Endocannabinoid-activated Nlrp3 inflammasome in infiltrating macrophages mediates β- cell loss in type 2 diabetes

CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions

(a) Significant biological processes (upper panel) and disease biomarkers (lower panel)

Postn MCM Smad2 fl/fl Postn MCM Smad3 fl/fl Postn MCM Smad2/3 fl/fl. Postn MCM. Tgfbr1/2 fl/fl TAC

Supplemental figure 1. PDGFRα is expressed dominantly by stromal cells surrounding mammary ducts and alveoli. A) IHC staining of PDGFRα in

Mitosis. Single Nano Micro Milli Macro. Primary. PCNA expression

Supplemental Information. Otic Mesenchyme Cells Regulate. Spiral Ganglion Axon Fasciculation. through a Pou3f4/EphA4 Signaling Pathway

SUPPLEMENTARY INFORMATION

Supplementary Figure 1

Supplementary Figure 1

Spatiotemporally regulated expression of transcription

SUPPLEMENTARY FIGURES

a) Primary cultures derived from the pancreas of an 11-week-old Pdx1-Cre; K-MADM-p53

Supplementary Figure 1: Neuregulin 1 increases the growth of mammary organoids compared to EGF. (a) Mammary epithelial cells were freshly isolated,

Supplementary Figure 1: si-craf but not si-braf sensitizes tumor cells to radiation.

Fig. S4. Current-voltage relations of iglurs. A-C: time courses of currents evoked by 100 ms pulses

Supplementary Table 1. Primer sequences for conventional RT-PCR on mouse islets

Supplementary Figure 1. Chimeric analysis of inner ears. (A-H) Chimeric inner ears with fluorescent ES cells and (I,J) Rainbow inner ears.

Nature Neuroscience: doi: /nn Supplementary Figure 1. Iliopsoas and quadratus lumborum motor neurons in the L2 spinal segment.

Polyclonal Secondary Antibodies

Ectopic pancreas formation in Hes1-knockout mice reveals plasticity of endodermal progenitors of the gut, bile duct, and pancreas

Supporting Information

Supplementary Figure 1

Erzsebet Kokovay, Susan Goderie, Yue Wang, Steve Lotz, Gang Lin, Yu Sun, Badrinath Roysam, Qin Shen,

Supplementary Figure 1

Diabetic pdx1-mutant zebrafish show conserved responses to nutrient overload and anti-glycemic treatment

Supplementary Information Supplementary Fig. 1. Elevated Usp9x in melanoma and NRAS mutant melanoma cells are dependent on NRAS for 3D growth.

Supporting Information

Inhibition of Cdk5 Promotes β-cell Differentiation from Ductal Progenitors

SUPPLEMENTARY INFORMATION

Supplemental Information. Tissue Myeloid Progenitors Differentiate. into Pericytes through TGF-b Signaling. in Developing Skin Vasculature

Supplementary Information. Induction of human pancreatic beta cell replication by inhibitors of dual specificity tyrosine regulated kinase

Developmental Biology

Supplementary Figure 1. A. Bar graph representing the expression levels of the 19 indicated genes in the microarrays analyses comparing human lung

Supplementary Information

Supplementary Materials for

Suppl Video: Tumor cells (green) and monocytes (white) are seeded on a confluent endothelial

Preferential reduction of β cells derived from Pax6 MafB pathway in MafB deficient mice

Supplementary Table 1. Characterization of HNSCC PDX models established at MSKCC

Figure S1 (A) Comparison of body weight and blood glucose levels in male wild-type (WT) and Cre littermates. Mice were maintained on a normal chow

Supplementary Figure S1: Tanycytes are restricted to the central/posterior hypothalamus

Supplemental Figure 1. Intracranial transduction of a modified ptomo lentiviral vector in the mouse

Supplementary Figure 1: Expression of Gli1-lacZ in E17.5 ovary and mesonephros. a,

Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell and patient-derived tumor organoids

Supplementary Figure 1

SUPPLEMENTARY INFORMATION

Supplemental Information

Supplementary Materials. for Garmy-Susini, et al, Integrin 4 1 signaling is required for lymphangiogenesis and tumor metastasis

Supplementary Figure 1

Supporting Information

Figure legends Supplemental Fig.1. Glucose-induced insulin secretion and insulin content of islets. Supplemental Fig. 2.

Tumor suppressor Spred2 interaction with LC3 promotes autophagosome maturation and induces autophagy-dependent cell death

Online Appendix Material and Methods: Pancreatic RNA isolation and quantitative real-time (q)rt-pcr. Mice were fasted overnight and killed 1 hour (h)

Supplemental Information Garmy-Susini, et al., PI3Kα activates integrin α4β1 to establish a metastatic niche in lymph nodes

CD31 5'-AGA GAC GGT CTT GTC GCA GT-3' 5 ' -TAC TGG GCT TCG AGA GCA GT-3'

Sox9: A Master Regulator of the Pancreatic Program. Philip A. Seymour

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

SUPPLEMENTARY INFORMATION

Quantification of early stage lesions for loss of p53 should be shown in the main figures.

In vitro differentiation optimization Flow Cytometry

IL-13 Augments Compressive Stress-induced Tissue Factor Expression in Human Airway Epithelial Cells

Islet viability assay and Glucose Stimulated Insulin Secretion assay RT-PCR and Western Blot

Supplementary Methods: Omalizumab Trial This double-blind, randomized, placebo-controlled trial was conducted at the University of Utah Hospital and

Supplemental Information. Induction of Expansion and Folding. in Human Cerebral Organoids

Supplementary Materials and Methods

Supplemental Materials, Nishimura et al, Page1 of 22 1

Acinar cells in the neonatal pancreas grow by self-duplication and not by neogenesis from duct cells Houbracken, Isabelle; Bouwens, Luc

Supplementary Figure 1 CD4 + T cells from PKC-θ null mice are defective in NF-κB activation during T cell receptor signaling. CD4 + T cells were

Supplementary Table 1. The primers used for quantitative RT-PCR. Gene name Forward (5 > 3 ) Reverse (5 > 3 )

Boucher et al NCOMMS B

Bone marrow-derived mesenchymal stem cells improve diabetes-induced cognitive impairment by

SUPPLEMENTARY DATA. Supplementary experimental procedures

Islets of Langerhans consist mostly of insulin-producing β cells. They will appear to be densely labeled

A Notch-dependent molecular circuitry initiates pancreatic endocrine and ductal cell differentiation

Zhu et al, page 1. Supplementary Figures

PKCζ Promotes Breast Cancer Invasion by Regulating Expression of E-cadherin and Zonula Occludens-1 (ZO-1) via NFκB-p65

A 10.8 kb ghr fragment containing exons 4 and 5 was PCR amplified with Pfu Turbo. DNA polymerase (Stratagene) and cloned into pcr Blunt II-TOPO vector

Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors

Supporting Information

Nature Neuroscience: doi: /nn Supplementary Figure 1. MADM labeling of thalamic clones.

SUPPLEMENTARY INFORMATION

Potentiation of Glucose-stimulated Insulin Secretion by the GPR40 PLC TRPC

Cell Therapy for Diabetes: Generating Functional Islets from Human Exocrine Tissue

Serafino et al. Thymosin α1 activates complement receptor-mediated phagocytosis in human monocyte-derived macrophages. SUPPLEMENTARY FIGURES

Pancreas-specific deletion of mouse Gata4 and Gata6 causes pancreatic agenesis

SUPPLEMENTARY INFORMATION

Supplementary Table; Supplementary Figures and legends S1-S21; Supplementary Materials and Methods

Biochemical and Biophysical Research Communications

DEVELOPMENT. Shelley B. Nelson, Ashleigh E. Schaffer and Maike Sander*

Transcription:

Impact of Sox9 Dosage and Hes1-mediated Notch Signaling in Controlling the Plasticity of Adult Pancreatic Duct Cells in Mice Shinichi Hosokawa 1,3,Kenichiro Furuyama 1,3, Masashi Horiguchi 1,3,Yoshiki Aoyama 1,3,Kunihiko Tsuboi 1,3,Morito Sakikubo 1,3 Toshihiko Goto 1,3,Koji Hirata 1,3,Wataru Tanabe 3,5,Yasuhiro Nakano 3, Haruhiko Akiyama 2, Ryoichiro Kageyama 4, Shinji Uemoto 1 and Yoshiya Kawaguchi 3,*

TM(-) Hes1 +/+ ;Rosa26R Hes1 f/f ;Rosa26R NICD;Rosa26R Supplementary Figure S1: Negative control experiment for Sox9 lineage-tracing study. Without tamoxifen treatment, X-gal positive cells are not detected in Hes1 +/+ ; Rosa26R, Hes1 loxp/loxp ; Rosa26R and Rosa NICD ; Rosa26R. Scale bars = 100 μm.

Hes1 f/f ;Rosa26R Hes1 +/+ ;Rosa26R A X-gal Hes1/DAPI X-gal/Hes1/DAPI Supplementary Figure S2 A. Hes1 expression was completely deleted in lineage-labeled duct cells. X-gal positive, lineage-labeled duct cells contain Hes1-expressing cells in Hes1 +/+ ; Rosa26R mice (upper panels), whereas in Hes1 loxp/loxp ; Rosa26R mice, Hes1 expression was not detected in lineage-labeled duct cells (white dotted lines in bottom panels). Note that Hes1-expressing cells were conserved in X-gal - ducts (bottom panels, yellow dotted lines) at the same frequency of that in wild type or Sox9 CreERT2 mice without tamoxifen treatment. Scale bars = 25 μm. B. Lineage-labeled acinar cells contain Notch-activated and non-activated cells in Rosa NICD ; Rosa26R mice. In Rosa NICD ; Rosa26R mice, X-gal positive acinar cells are composed of EGFPpositive and negative populations, the former indicating successful recombination of both Rosa26R and Rosa NICD-IRES-EGFP alleles (compare the absence of X-gal and EGFP-double positive cells in the upper and middle panels). Activated Notch signaling by NICD induction was confirmed by X-gal, Hes1 double positive acinar cells (bottom panels). Yellow arrows indicate EGFP + /X-gal + or Hes1 + /X-gal + acinar cells. White arrows indicate EGFP - /X-gal + or Hes1 - /X-gal + acinar cells. Scale bars = 25 μm.

NICD;Rosa26R NICD;Rosa26R Hes1 +/+ ;Rosa26R B X-gal EGFP X-gal/EGFP/DAPI X-gal Hes1 X-gal/Hes1/DAPI

1year 2months 1month A Hes1 f/+ ;Rosa26R Hes1 f/f ;Rosa26R B X-gal Ins/Glu/DAPI islet islet islet Supplementary Figure S3: Hes1 is dispensable for the maintenance of progenitor population. (A) Long-term chase at 1 month, 2 months and one year after single tamoxifen injection. As time proceeds, lineage-labeled acinar cells gradually expanded in number in both Hes1 loxp/+ ; Rosa26R and Hes1 loxp/loxp ; Rosa26R mice (representative pictures are shown). Notably, even one year after tamoxifen injection, pancreatic duct cells retain their X-gal labeled status and X-gal positive acinar cells are broadly spread, suggesting that Hes1 is dispensable for the self-duplication of Sox9-expressing progenitor cells and differentiation into the acinar cell type. (B) No X-gal-labeled cells are observed in insulin+ (green) or glucagon+ (red) endocrine cells even in the long-term chase periods. Scale bars = 100 μm.

Hes1/CK19/DAPI A TM TM TM TM analysis 0 2 4 6 7 B Hes1 +/+ ;Rosa26R Hes1 f/f ;Rosa26R NICD;Rosa26R Supplementary Figure S4: Modulation of Notch activity by high-dose tamoxifen treatment. (A) To get more efficient recombination, tamoxifen was injected four times. (B) High dose tamoxifen treatment resulted in a decrease and increase in the number of Hes1-expressing cells in Hes1 loxp/loxp ; Rosa26R and Rosa NICD ; Rosa26R mice, respectively. White and yellow arrowheads indicate Hes1 + duct/centroacinar and Hes1 + acinar cells, respectively. Scale bars = 50 μm.

NICD;Rosa26R Hes1 +/+ ;Rosa26R Hes1 Ptf1a Hes1/Ptf1a/DAPI HE Supplementary Figure S5: Emergence of Hes1/Ptf1a double positive acinar cells by NICD induction. In Rosa NICD ; Rosa26R mice (lower panels), acinar cells contain Hes1/Ptf1a double positive cells (yellow arrows) which were not detected in the control mice (upper panels). Scale bars = 25 μm.

Supplementary Table1 The ratio of Sox9 positive cells among all epithelial cells (% of total DAPI + cells) Genotype Wild type P1 n % of total epithelial cells 3 13.3±0.23 (Sox9 + / DAPI + : 2634/19707) Sox9 CreERT2 P1 3 12.7±0.97 (Sox9 + / DAPI + : 2690/21756) Wild type adult 3 9.11±0.22 (Sox9 + / DAPI + : 8390/92352) Sox9 CreERT2 adult 3 9.22±0.68 (Sox9 + / DAPI + : 8138/88064) The numbers shown represent the means number ± standard error of the means(s.e.m)

Supplementary Table2 Hes1 co-localization among CK19 + ductal population (% of total CK19 + cells counted) Adult pancreas Genotype n % of total duct cells Wild type 3 62.1±1.62 (Hes1 + CK19 + / total CK19 + : 647 /1122 ) Sox9 CreERT2 3 63.6±1.57 (Hes1 + CK19 + / total CK19 + : 693 /1084 ) The numbers shown represent the means number ± standard error of the means(s.e.m)

Supplementary Table3 Number of lineage labeled acinar cells counted day 3 Genotype Hes1 +/+ ;Rosa26R Hes1 +/+ ;Rosa26R Hes1 +/+ ;Rosa26R Hes1 loxp/loxp ;Rosa26R Hes1 loxp/loxp ;Rosa26R Hes1 loxp/loxp ;Rosa26R Hes1 loxp/loxp ;Rosa26R Hes1 loxp/+ ;Rosa26R Hes1 loxp/+ ;Rosa26R Hes1 loxp/+ ;Rosa26R Hes1 loxp/+ ;Rosa26R Rosa NICD ;Rosa26R Rosa NICD ;Rosa26R Rosa NICD ;Rosa26R Rosa NICD ;Rosa26R day 10 X-gal + acinar cells/20fields 664 609 511 709 1188 1250 1413 332 520 531 588 220 510 355 146 average 594 1140 492 308 Hes1 +/+ ;Rosa26R 1811 Hes1 +/+ ;Rosa26R 1913 Hes1 +/+ ;Rosa26R 1231 Hes1 +/+ ;Rosa26R 2011 Hes1 loxp/loxp ;Rosa26R 2800 Hes1 loxp/loxp ;Rosa26R 3402 Hes1 loxp/loxp ;Rosa26R 2393 Hes1 loxp/loxp ;Rosa26R 2841 Hes1 loxp/+ ;Rosa26R 779 Hes1 loxp/+ ;Rosa26R 2262 Hes1 loxp/+ ;Rosa26R 2035 Hes1 loxp/+ ;Rosa26R 1131 Rosa NICD ;Rosa26R 1138 Rosa NICD ;Rosa26R 672 Rosa NICD ;Rosa26R 774 Rosa NICD ;Rosa26R 846 1741 2859 1551 857

Supplementary Table4 The ratio of Sox9 positive cells among all epithelial cells (% of total DAPI + cells) Adult pancreas Genotype Sox9 CreERT2 3 n % of total epithelial cells 9.39±0.83 (Sox9 + / DAPI + : 7268/75825) Rosa NICD 3 9.18±0.54 (Sox9 + / DAPI + : 9118/99306) Hes1 loxp/loxp 3 8.09±0.17 (Sox9 + / DAPI + : 7938/98487) The numbers shown represent the means number ± standard error of the means(s.e.m)

Supplementary Table 5 Primary Antibodies Antigen Species Dilution Supplier Amylase Amylase CK19 Glucagon Rabbit Goat Rat Rabbit 1:1000 1:100 1:500 1:400 Sigma Santa Cruz DSHB DAKO GFP Chicken 1:500 abcam Hes1 Insulin PECAM-1 Ptf1a Sox9 Sox9 Rabbit Guinea Pig Rat Rabbit Rabbit Goat 1:5000 1:400 1:20 1:10000 1:1000 1:50 Dr.Sudo DAKO BD Biosciences Dr.Hoshino Millipore Santa Cruz

Supplementary Table 6 Secondary Antibodies Conjugate Antigen Species Dilution Supplier Alexa Fluor488 Mouse IgG Goat 1:1000 Molecular Probes Alexa Fluor488 Rabbit IgG Donkey 1:1000 Molecular Probes Alexa Fluor488 Guinea pig IgG Goat 1:1000 Molecular Probes Alexa Fluor488 Goat IgG Donkey 1:1000 Molecular Probes Cy3 Rat IgG Donkey 1:500 Chemicon Cy3 Rabbit IgG Donkey 1:500 Chemicon

2024 © healthdocbox.com Privacy Policy | Terms of Service | Feedback