Organogenesis II:
Endodermal Organs
Major Derivatives of the Germ Layers
Epidermis, CNS, PNS, Melanocytes
Gut, Lungs, Liver, Pancreas,
Gall Bladder, Thymus, Thyroid…
Notocord, Muscle, Bone, Dermis, Heart, Kidney,
Somatic Gonad, Vaculature, Blood
Human Embryo
Endodermal Derivatives
A/P Patterning of the Endoderm
Genes expressed in endoderm
Grapin-Botton and Melton
Endoderm is also patterned by interactions with neighboring mesoderm
Therefore HOX code of mesoderm also likely to be important for endoderm
An Example of Endoderm Organogenesis: The Pancreas
High
Blood
Sugar
Low
Blood
Sugar
Insulin
Glucagon
Import Glucose Gluconeogenesis
Into Tissues
Exocrine
Endocrine
Islets of
Langerhans
Glucagon
Insulin
Somatostatin
Pancreatic
Polypeptide
An estimated 18 million people suffer from Type I or II Diabetes
A/P Patterning of the Endoderm
Genes expressed in endoderm
Grapin-Botton and Melton
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
Epithelial-mesenchymal Interactions
The Mesoderm Induces Pancreas Formation
pdx1
Kim and MacDonald
Glucagon
Control
Notochord
Removed
Insulin
Epithelial-mesenchymal Interactions
The Mesoderm Induces Pancreas Formation
pdx1
1) The notochord allows dorsal endoderm to form dorsal pancreas
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
Regulation of Pancreatic Differentiation by Signals from Blood Vessels
Science, 2001
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
Surgical ablation of aorta causes defect
in pancreas development (Xenopus)
Epithelial-mesenchymal Interactions
The Mesoderm Induces Pancreas Formation
pdx1
1) The notochord allows dorsal endoderm to form dorsal pancreas
2) The dorsal aorta maintains the dorsal pancreas
Specifying Ventral Pancreas vs. Liver
V
D
(pancreas)
(liver)
Ventral Endoderm
Forms Pancreas When
Cultured in Isolation
Cardiac Mesoderm Inhibits
Pancreas (and allows Liver
Development)
(Cardiac Mesoderm-Ventral
Endoderm Co-culture)
FGF2 Promotes
Liver over Pancreas
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?
Epithelial-mesenchymal Interactions
The Mesoderm Induces Pancreas Formation
pdx1
1) The notochord allows dorsal endoderm to form dorsal pancreas
2) The dorsal aorta maintains the dorsal pancreas
3) Cardiac mesoderm promotes liver over ventral pancreas (default?)
Pdx1 (Insulin promoter factor 1, IPF1) is
used at multiple steps of pancreas
development
How do we know this?
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
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
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
Cell-type specification in the pancreas
Morphogenesis of the pancreas: exocrine vs. ductal system
Tube Morphogenesis
Lung
Vasculature
Kidney
Pancreas
Mammary Glands
Salivary Glands
Drosophila Trachea
Morphogenesis of the pancreas: Islet (endocrine) organization
Endocrine
Islets of
Langerhans
Glucagon
Insulin
wt
Somatostatin
Pancreatic
Polypeptide
DN-E-cad
PMID: 8787762
Cell-type specification in the pancreas
Ptf1a
Multipotent
Pancreatic
Progenitor
Cells (MPCs)
Acinar (exocrine)
Ngn3
Nkx6
Ductal/Endocrine
Notch (Lateral Inhibition)
promotes MCP vs. Differentiation
Notch (Lateral Inhibition)
promotes Duct vs. Endocrine
Ducts
Ngn3 is required for exocrine pancreas development
PMID: 10677506
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
Stage-dependent induction of different endocrine cell types by Ngn3
PMID:17336910
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
Dox Repressible pdx-1
An example protocol for human ES cells
PMID: 18288110
Can Functioning Beta Cells Be Derived from ES or ips Cells for Therapy?
(and other groups)
Derived cells exhibit glucose-responsive insulin release
Transplantation into mouse diabetes model corrects high blood
glucose levels and allows for better response to glucose
challenge (not shown)