Branching morphogenesis in the embryonic pancreas

1
Branching morphogenesis in the embryonic pancreas

• Anna Means, PhD
• Division of Surgical Oncology
• Dept. of Cell and Developmental Biology
• Vanderbilt University Medical Center

2
Pancreatic development proceeds in stages
1st Wave of differentiation
2nd Wave of differentiation
Endocrine and exocrine differentiation
specification
budding
branching
E9.5
E12.5
E10.5
E8.5
E14.5 E18.5
3
The pancreas is well studied in terms of

• Specification
• Differentiation
• Progenitor status
• Survival/proliferation

But there has been little work on branching
morphogenesis
4
Early pancreatic development
lobulation
budding
branching
evagination
5
Early branching and differentiation
E11.5
Glucagon/
Pdx1
6
Early branching and differentiation
E12.5
E11.5
Glucagon/
Pdx1
7
Early branching and differentiation
E13.5
E12.5
E11.5
Glucagon/
Pdx1
8
Step 1 budding
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Morphogenesis by cell shape and movement
Wessels and Evans, 1967
Means
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Step 2 lobulation
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Salivary gland initiates branching by cell
contraction
Cleft formation
Microfilaments contract on one side
Microfilament contraction
Tissue bends
Cytochalasin B
Wash out Cytochalasin B
Spooner and Wessells
12
Salivary gland initiates branching by cell
contraction
Cleft formation
Microfilaments contract on one side
Microfilament contraction
Tissue bends
Cytochalasin B
Wash out Cytochalasin B
Spooner and Wessells
13
Pancreas is also lobulated just before branching
begins
14
ECM components are deposited differentially in
clefts vs. lobules
Salivary gland
Fibronectin RNA
Fibronectin ISH
Perlican
/
Sakai et al., 2003
15
Blocking fibronectin inhibits lobulation in
salivary gland
Buds/gland
control
a-FN
a-FN
Sakai et al., 2003
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Blocking fibronectin expression inhibits
branching in salivary gland
Sakai et al., 2003
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Adding fibronectin to media enhances branching
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Fibronectin from media gets deposited in forming
clefts
Design
Red-FN
2 h
Green-FN
4 h
Visualize
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Step 3 branching morphogenesis
20
Cell-autonomous and non-cell autonomous
regulation

• Cell autonomous regulators
• Pdx1
• Ptf1a
• Non-cell autonomous regulators
• Growth factors
• ECM components

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Cell-autonomous regulation
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Pdx1 is a homeodomain transcription factor that
is required for multiple aspects of pancreatic
development
23
Loss of Pdx1 severely limits size and lobulation
Pdx1/-
Pdx1/-
Pdx1-/-
Means, Wright
24
Loss of Pdx1 inhibits branching
Pdx1/-
Pdx1-/-
E12.5 mouse pancreatic anlage
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Ptf1a is a transcription factor expressed in
pancreatic progenitors
Kawaguchi, 2002
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Loss of Ptf1a inhibits branching(along with
other processes)
Kawaguchi, 2002
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How can transcription factors regulate branching?
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Non-cell-autonomous regulation
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Mesenchyme is required for pancreatic branching
Golosow and Grobstein, 1962
No mesenchyme
Mesenchyme
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So what is the mesenchymal signal?
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Mesenchymal players

• Factors that maintain mesenchyme
• Factors that signal to epithelium

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Isl1 is a transcription factor required for
mesenchymal survival
NO BRANCHING
Isl1
Loss of Isl1 means loss of mesenchyme and
therefore no branching!
33
Fgf10 is expressed in pancreatic mesenchyme
Fgf10 ISH
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Loss of Fgf10 decreases size of pancreas
Bhushan
WT
Fgf10-/-
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Loss of the Fgf10 receptor decreases branching
E14.5
Otonkoski
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Fgf10 is required to maintain Pdx1 expression
Pdx1 expression
Bhushan
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EGFR regulation of branchingepithelial or
mesenchymal?
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EGFR and ErbB4 line the boundary between
epithelium and mesenchyme
EGFR/
Glucagon
E12.5
2X
ErbB4/
Glucagon
2X
39
Egfr RNA is widely distributed in and around the
developing pancreas
sense
as
In situ hybridization
as
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Pharmacological inhibition of EGFR signaling
inhibits branching, promotes early differentiation
Control culture
6 day
2 day
EKI treated
10 nM EKI-785 (EGFR inhibitor)
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Egfr null mutation reduces size and branching
Egfr/
Egfr-/-
1/8 volume
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EGFR/ErbB4 signaling promotes mesenchymal
survival in culture
HB-EGF
control
Day 6
Mesenchyme Alone
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Overexpression of sHB-EGF alters pancreatic
mesenchyme
Pdx1-sHBEGF transgene
WT
E14.5 pancreas
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Overexpression of sHB-EGF alters pancreatic
mesenchyme
Pdx1-sHBEGF
WT
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Mammary gland ductal branching requires EGFR in
mesenchyme
Wiesen et al., 1999
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Mammary gland ductal branching requires EGFR in
mesenchyme
Wiesen et al., 1999
47
Cell-cell contacts that regulate epithelial
branching
48
Epimorphen regulates branching in absence of
mesenchyme
Anti-EPM ab.
Gittes
49
What is epimorphen?

• Also called syntaxin-2
• Membrane associated protein
• On cytoplasmic face, EPM is part of SNARE complex
  involved in exocytosis
• On outer face, EPM regulates branching of many
  organs

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Laminin regulates branching in absence of
mesenchyme
a-laminin
Ab. control
Neutralized Ab.
a- a6 integrin
Gittes
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How can ECM proteins regulate branching?
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Mesodermal signals in pancreatic development
Mesoderm
Endoderm
Shh
activin
notochord
RA
Pdx1
aorta
Ptf1a
Isl-1
RA
EGFR?
Fgf10
Splanchnic mesenchyme
Growth/branching
53
Mesodermal signals in pancreatic development
Mesoderm
Endoderm
Shh
activin
notochord
RA
Pdx1
aorta
Loss of mesenchyme
Ptf1a
Isl-1
RA
EGFR?
Fgf10
Splanchnic mesenchyme
Growth/branching
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Mesodermal signals in pancreatic development
Mesoderm
Endoderm
Shh
activin
notochord
RA
Pdx1
aorta
Loss of mesenchyme
Ptf1a
Isl-1
RA
EGFR?
Fgf10
Splanchnic mesenchyme
Growth/branching
Loss of epithelial progenitor cells
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Mesodermal signals inducing branching
Mesoderm
Endoderm
ECM/integrin signaling is another critical
component of branching
As Lindsay Hinck showed, adhesion is a critical
part of branching morphogenesis!