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Organogenesis II: Endodermal Organs

Organogenesis II: Endodermal Organs. Gut, Lungs, Liver, Pancreas, Gall Bladder, Thymus, Thyroid…. Epidermis, CNS, PNS, Melanocytes. Notocord, Muscle, Bone, Dermis, Heart, Kidney, Somatic Gonad, Vaculature, Blood. Major Derivatives of the Germ Layers. Human Embryo. Endodermal Derivatives.

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Organogenesis II: Endodermal Organs

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  1. Organogenesis II:Endodermal Organs

  2. Gut, Lungs, Liver, Pancreas, Gall Bladder, Thymus, Thyroid… Epidermis, CNS, PNS, Melanocytes Notocord, Muscle, Bone, Dermis, Heart, Kidney, Somatic Gonad, Vaculature, Blood Major Derivatives of the Germ Layers

  3. Human Embryo

  4. Endodermal Derivatives

  5. A/P Patterning of the Endoderm Grapin-Botton and Melton Genes expressed in endoderm Endoderm is also patterned by interactions with neighboring mesoderm Therefore HOX code of mesoderm also likely to be important for endoderm

  6. High Blood Sugar Low Blood Sugar Insulin Glucagon Import Glucose Into Tissues Gluconeogenesis Exocrine Endocrine Islets of Langerhans Glucagon Somatostatin Insulin Pancreatic Polypeptide An Example of Endoderm Organogenesis: The Pancreas An estimated 18 million people suffer from Type I or II Diabetes

  7. A/P Patterning of the Endoderm Grapin-Botton and Melton Genes expressed in endoderm

  8. PDX-1/Ipf-1 is Essential for Pancreas Development The pancreas (p) is missing in pdx mutants In pdx- (knock in) animals: -pdx-lacZ is still expressed -a pancreatic bud forms -no further pancreatic development is observed

  9. Epithelial-mesenchymal Interactions The Mesoderm Induces Pancreas Formation pdx1 Kim and MacDonald

  10. Glucagon Insulin Control Notochord Removed

  11. Epithelial-mesenchymal Interactions The Mesoderm Induces Pancreas Formation pdx1 1) The notochord allows dorsal endoderm to form dorsal pancreas

  12. Signals From the Notochord Pattern All Three Germ Layers 4, 6,7 D/V Patterning of Neural Tube (Ectoderm)D/V Patterning of Somite (Mesoderm)Pancreas Primordium (Endoderm

  13. Surgical ablation of aorta causes defect in pancreas development (Xenopus) Science, 2001 Regulation of Pancreatic Differentiation by Signals from Blood Vessels Pdx1 expression originates adjacent to dorsal aorta and ventral veins (mouse) Genetic ablation of vessels (Yoshitomi and Zaret 2004) shows that dorsal aorta is essential for maintenance of dorsal pancreas, but are not important for ventral pancreas

  14. Epithelial-mesenchymal Interactions The Mesoderm Induces Pancreas Formation pdx1 The notochord allows dorsal endoderm to form dorsal pancreas The dorsal aorta maintains the dorsal pancreas

  15. Specifying Ventral Pancreas vs. Liver V D

  16. FGF2 Promotes Liver over Pancreas (pancreas) (liver) Ventral Endoderm Forms Pancreas When Cultured in Isolation Cardiac Mesoderm Inhibits Pancreas (and allows Liver Development) (Cardiac Mesoderm-Ventral Endoderm Co-culture)

  17. Model: Liver and Pancreas arise from a bipotential primordium FGF’s from Cardiac Mesoderm promote liver over pancreas Experimental manipulations can induce liver to pancreas or pancreas to liver transformations--useful for diabetes therapy?

  18. Epithelial-mesenchymal Interactions The Mesoderm Induces Pancreas Formation pdx1 The notochord allows dorsal endoderm to form dorsal pancreas The dorsal aorta maintains the dorsal pancreas Cardiac mesoderm promotes liver over ventral pancreas (default?)

  19. Pdx1 (Insulin promoter factor 1, IPF1) is used at multiple steps of pancreas development How do we know this?

  20. Engineering a Conditional Allele: Conditional Expression Using the Tet ON/OFF System H. Bujard and colleagues tTA: Dox repressed txn factor rtTA: Dox induced txn factor tet repressor (tetR) = tetracycline-responsive DNA binding protein from tetracycline resistance operon-- only binds DNA in ABSENCE of tetracyclines such as doxycycline (Dox) Reverse tetR (rtetR) = mutant form of tetR that now only binds DNA in PRESENCE of Dox VP16 = Powerful mammalian transcriptional activation domain

  21. Goal: Make conditional allele of Pdx-1 (Dox responsive) Holland, PNAS 2002 Original Pdx-1 locus tTA “knocked into” Pdx-1 locus: 1) makes mutant for Pdx-1 2)expresses tTA in Pdx-1 pattern tTA-responsive Pdx-1 transgene Also expresses GFP when expressing Pdx-1

  22. Pdx-1 (Insulin promoter factor 1, IPF1) is used at multiple steps of pancreas development Dox repressible allele of Pdx-1: Dox throughout gestation: Looks like Pdx-1 null Add Dox at midpoint of pancreas development: Block further development Give Dox to adults: Decreased insulin and glucose regulation Therefore, Pdx-1 is required throughout pancrease development and in adult pancreas

  23. Cell-type specification in the pancreas

  24. Morphogenesis of the pancreas: exocrine vs. ductal system

  25. Tube Morphogenesis Lung Vasculature Kidney Pancreas Mammary Glands Salivary Glands Drosophila Trachea

  26. Endocrine Islets of Langerhans Glucagon Somatostatin Insulin Pancreatic Polypeptide Morphogenesis of the pancreas: Islet (endocrine) organization wt DN-E-cad PMID: 8787762

  27. Cell-type specification in the pancreas Ptf1a Acinar (exocrine) Multipotent Pancreatic Progenitor Cells (MPCs) Ngn3 Notch (Lateral Inhibition) promotes Duct vs. Endocrine Ductal/Endocrine Nkx6 Notch (Lateral Inhibition) promotes MCP vs. Differentiation Ducts

  28. Ngn3 is required for exocrine pancreas development PMID: 10677506

  29. Ngn3-positive cells are unipotent in terms of endocrine cell type -ngn3 clones small—usually one cell. Therefore ngn3+ cells are not multipotent “stem” cell. -When multicellular, ngn3 clones usually give rise to single type of endocrine cell PMID:19793886

  30. Stage-dependent induction of different endocrine cell types by Ngn3 PMID:17336910

  31. Pancreas Regeneration Lineage analysis using cell-type specific and drug inducible CRE ß-cell specific CRE-ER (CRE activivated by tamoxifen) CRE induces AP expression Permanent record of CRE activity at time of tamoxifen treatment -Give “pulse” of tamoxifen to label subset of ß-cells with AP -Count % of labeled ß-cells -Ressect pancreas to induced regeneration -See if % labeled ß-cells changes If new ß-cells come from undifferentiated progenitor which doesn’t express insulin, new ß-cells won’t be labeled If new ß-cells come from old ß-cells, then some new ß-cells with be labeled (% labeled ß-cells won’t change) Result: % of labeled ß-cells doesn’t change after regereration Therefore old ß-cells, or at least differentiated, insulin expressing cells, give rise to new ß-cells Doesn’t support undifferentiated adult stem cell model

  32. Dox Repressible pdx-1 An example protocol for human ES cells PMID: 18288110

  33. Can Functioning Beta Cells Be Derived from ES or ips Cells for Therapy? (and other groups) Transplantation into mouse diabetes model corrects high blood glucose levels and allows for better response to glucose challenge (not shown) Derived cells exhibit glucose-responsive insulin release

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