Analysis of the plasticity of duct cells in the embryonic and adult pancreas. Myriam Solar Hospital Clinic de Barcelona Barcelona 2009
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1 Analysis of the plasticity of duct cells in the embryonic and adult pancreas Myriam Solar Hospital Clinic de Barcelona Barcelona 2009
2 Major periods of formation of new β-cells: 1- Embryogenesis (secondary transition) 2- Postnatal life Neonatal period Long-term growth Regeneration in response to injury
3 Hnf1β is a specific marker for primitive and adult pancreatic duct epithelium
4 Adult Generation of a BAC transgenic to trace the lineage of pancreatic ducts 7.1 Kb Wild type Hnf1 b CreER; Rosa26R - 4 OHT + 4 OHT Targeting vector Promoter CreER IRES EGFP polya Neo Intron1 Cre RP23-304H7 (192 Kb) NotI exon1 101 Kb 36 Kb Hnf1b locus 55Kb NotI Embryo Transgenic BAC (196.7 Kb) NotI Promoter CreER IRES EGFP polya 4.7 Kb Intron1 NotI Cre / CK19 Cre Hnf1 b CreER Hnf1 β CreER X Rosa26 STOP LacZ Rosa26R + Tamoxifen Hnf1 b CreER/ Rosa26R Hnf1 β CreER Rosa26 LacZ
5 At E12.5-E13.5 CreER is expressed (like Hnf1b) in duct-like trunk cells of the developing pancreas Cre/E-Cad E12.5-noTAM E hTAM
6 4-OHT E12.5 E18.5 Adult Acinar? Ductal HNF 1b?? Endocrine E12.5-E13.5 E18.5 or adult
7 Hnf1β+ cells from E12.5-E13.5 give rise to differentiated duct, acinar, and endocrine cells Bgal/Amyl Acinar Ductal Bgal/CK19 HNF 1b Endocrine Bgal/Ins Bgal/Glu E12.5-E13.5 E18.5 or adult
8 During the secondary transition (E13.5-E16.5) CreER is expressed in the ductal epithelium Cre/Ngn3 100 Cre/InsGlu % of cells with CreER ND E14.5 Hnf1b Ins Gluc Cpa1 Ngn3
9 4-OHT E13.5 E14.5 E18.5 Adult Acinar? Ductal HNF 1b?? Endocrine E13.5-E16.5 E18.5 or adult
10 4-OHT E13.5 E14.5 E18.5 Adult E14.5 Bgal/Hnf1b E18.5 Bgal/Hnf1b 5% Ductal (Hnf1b+) Non-ductal (Hnf1b-) 50% 50% 95% Hnf1b+ duct cells of the secondary transition give rise to non-duct cells
11 Duct 4-OHT cells from the secondary transition give rise to differentiated duct and endocrine cells E13.5 E14.5 E18.5 Adult Bgal/CPA Acinar Ductal Bgal/Sox9 %Colocalization with Cre E14.5 ND 0 Hnf1b Ins Gluc Cpa1 Ngn3 HNF1b+ Endocrine Bgal/Ins Bgal labelling rate E18.5 E14.5 ND Hnf1b Ins Gluc Cpa1 Ngn3 E13.5-E16.5 E18.5 or adult
12 At birth CreER remains expressed broadly throughout the ductal epithelium Cre/CK19 Cre/Ins Cre/Glu
13 4-OHT Acinar E18.5 P0 P14? Ductal HNF 1b?? Endocrine E18.5 P14
14 4-OHT HNF 1b E18.5 P0 P14 Bgal/CPA Acinar Ductal Endocrine E18.5 P14 Bgal/CK19 Bgal/Ins 100 % Coloc. Cre Bgal labelling rate P0 ND CK19 Ins Gluc Aci P14 ND CK19 Ins Gluc Aci
15 Pancreatic duct cells primarily give rise to duct cells during the neonatal period. HNF 1b Acinar Ductal E18.5 P14 Bgal/CPA Bgal/CK19 Bgal/Ins 100 % Coloc. Cre Bgal labelling rate P0 ND CK19 Ins Gluc Aci P14 ND CK19 Ins Gluc Aci
16 Pancreatic 4-OHT duct cells primarily give rise to duct cells during adult life HNF 1b Young adults Young adult 1 week 6 months Acinar Bgal/CPA 1,2 Ductal 6m after T Bgal/CK19 Bgal/Ins Bgal labelling rate Bgal labelling rate 1 0,6 0,2 0 Hnf1b Ins Glu Exo 1,2 1 0,6 0,2 Young adults ND 6 m after T ND 0 Hnf1b Ins Glu Exo
17 Do new beta-cells that arise during regeneration originate from the pancreatic duct epithelium?
18 Hnf1β and Hnf1bCreER are widely expressed in the ductal epithelium, including main, interlobular, intralobular, intercalated, and centroacinar cells X-GAL in head, adult Tam-treated Hnf1bCreER-R26R Anti-HNF1β DAB Cre/CK19 Intrerlobular duct Main duct Centroacinar cells
19 4-OHT PDL or Alloxan/EGF-gastrin Young adult 1 week 4 weeks Acinar? Ductal HNF 1b?? Endocrine 4 weeks of age Post-regeneration
20 β-gal expression remains in the pancreatic duct epithelium of the ligated pancreas Acinar Bgal/CK/Ins Ductal HNF 1b Bgal/CK/Glu Endocrine 4 weeks of age Post-regeneration
21 PDL causes a doubling of beta-cell mass Hnf1bCreER / R26R X-gal in beta-cells does not increase after PDL Hnf1bCreER / R26R Labelling efficiency in Cyotoleratin+ or Hnf1b+ cells: 30-54%
22 Tubular complexes formed after pancreatic duct ligation are derived from pre-existing duct cells
23 Before E13.5 Early morphogenesis ~E13.5 E16.5 Secondary transition After E18.5 Physiological & regenerative growth Acinar HNF 1b HNF 1b HNF 1b Duct Endocrine
24 Aknowledgements Hospital Clinic/IDIBAPS Mercè Martín Carina Cardalda Miguel Ángel Maestro Vanessa Grau Jorge Ferrer Vrije Universiteit Brussel Nele de Medts Isabelle Houbracken Xiaobo Xu Harry Heimberg Luc Bouwens UAB transgenic core facility Anna Pujol Reagents Mike German Pedro Herrera
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