Stem Cells and Lineages: Mechanisms of
Lung Development
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Overview of stages of lung development
Epitheial-Mesenchymal interactions
Budding
Branching morphogenesis
Sacularization and Alveolarization (Maturation)
Genes and growth factors that control these
processes
Focus on how these molecules were shown to play
a role in the processes
Stem Cells and Lineages: Mechanisms of
Lung Development
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Lungs did not develop from gills, they are a
completely separate structrure
Developed from swim bladders in fish
Gills develop from pharyngeal arches
independently of lungs.
Bladder
Early
Late
Review
of mouse
lunglung
development
Overview
of mouse
development
Canalicular
Pseudoglandul
ar
Saccular
Alveolar
Cordoso, Dev. Dyn. 219:121 (2000)
Neonatology: SE Wert
5th edition 1999.
Overview
of mouse
development
Review
of mouse
lung lung
development
Canalicular
Pseudoglandul
ar
Saccular
Alveolar
Cordoso, Dev. Dyn. 219:121 (2000)
Neonatology: SE Wert
5th edition 1999.
Summary of stages
Nodal (TGF member) Foxa2, Sox17,
GATA4,6, Stat3 needed for gut tube
formation as shown by KOs
Bladder
Early
Late
Budding
Evidence for players
1) Tissue recombination experiments (70 years ago to today)
2) In situ hybridization (guilt by assocation)
3) Gene KOs and transgenics
4) In vitro culture assays with beads and factors
1) Tissue recombination experiments (70 years ago to today)
Rudnick, D. 1933 Chick lung rudiments transplanted
onto Chorioallantoic membranes and cultured.
Mesenchyme required for branching.
Wessells, N 1970 Lung buds form opposite sites where
lung mesenchyme is placed in vitro.
Masters, JRW 1976 Mesenchyme is needed to induce
branching morphogenesis in vitro from tracheal buds.
Shannon, JM 1994 Distal mesenchyme can induce bud
and branching of proximal trachea
Shannon, JM 1994 Distal mesenchyme can induce
bud and branching of proximal trachea
Shannon, JM 1994 Distal mesenchyme can induce
bud and branching of proximal trachea
Day 13 lung
Grafted Trachea time 0 Grafted Trachea 24hrs
Shannon, JM 1994 Distal mesenchyme can induce
bud and branching of proximal trachea
Shannon, JM 1994 Distal mesenchyme can induce
bud and branching of proximal trachea
Get relatively normal looking lung lobes when
mesenchyme is grafted next to trachea.
What are inducing molecules from normal E99.5 gut mesenchyme that induce budding?
FGF10 is believed to be the primary bud
inducing molecule. Retinoic acid receptors
are also implicated in initial lung formation as
double KOs of 2 prevent trachea formation.
Evidence for FGF10?
1) Is expressed in mesenchyme opposite
where lung bud forms.
2) KO of FGF10 prevents budding and lung
formation.
Little else known about bud formation
How is FGF10 localized? What target genes
are there in the epithelium that respond
to FGF10?
Branching morphogenesis
• FGF10, TTF-1, Shh other molecules in
a repeating signalling network during
branching
• Evidence primarily from KOs and
explant studies
Branching morphogenesis
Foxa1 or Foxa2 needed for Shh
expression in epithelium
FGF9-Shh network
also implicated in
early branching. KO
reduces branching.
Development 133, 1507-1517 (2006)
doi:10.1242/dev.02313
Branching lineage
Genetic control of branching lineage
Spry2 (Sprouty 2) a member
of
a
tyrosine
kinase
inhibitor family.
Ectopic
domain
branching
in
Spry22/2 mutants. a, The
RCd lobe (ventral view) of
an E12.5 control (Spry21/2)
lung
with
a
single
secondary
branch
(V1,
circled) off RCd, at the level
of RCd.L4 (L4). Below,
RCd.V1
lineage
and
schematic of RCd with a
single ventral secondary
branch (V1). b, Same view of
an E12.5 Spry22/2 lung
showing the normal ventral
secondary branch (V1) and
an ectopic branch (V*) that
forms earlier and proximal
to V1. V* has already
sprouted
additional
generations of branches.
Below,
lineage
and
schematic show V* plus
additional ectopic ventral
branches (V**, V***; dashed
lines in schematic) seen in
other
Spry22/2
lungs
(Supplementary
Fig.4).
Scale bar (for a and b), 200
mm.
Genetic control of branching
• Shh (Epith.), Hip1, Ptc1,Gli2, Gli3 (mesen.)
• FGF10, FGF9 (mesen.)
• TTF-1 aka Titf1 (Epith.)
• Foxa1+Foxa2, single KOs branch normally req. for Shh
expression in epith.
• BMP4 (Tgf family) (mesen.)
• Pod1 (bHLH TF in mesen.)
• -catenin signalling in epith. And mesen.
• Foxf1 (mesen.)
• Tbx4, 5 req. for Fgf10 express. in mesen. (siRNA studies)
• Hoxa5 (mesen.)
• Spry2 FGF tyrosine kinase inhibitor (Epith.)
Genetic control of branching
Reciprocal Epithelial-Mesenchymal
interactions mediated by growth factor- and
cell contact-regulated transcriptional networks.
These interactions both constitute a program
of growth, morphogenesis and differentiation
and then help establish the quazi-stable
epigenetic states that characterize each
differentiated cell.
Overview
of mouse
development
Review
of mouse
lung lung
development
Canalicular
Pseudoglandul
ar
Saccular
Alveolar
Cordoso, Dev. Dyn. 219:121 (2000)
Neonatology: SE Wert
5th edition 1999.
Canalicular and saccular phases
(later maturation E16.5-P5)
• Transition from undifferentiated to fully
differentiated cells and marked
reduction in cell proliferation.
• KOs, conditional KOs and conditional
transgenics
• In vitro culturing of lungs
Canalicular phase (E16.5-17.5)
what cell types are formed in conducting airways?
Foxj1 KO
eliminates
cilia on
cells
p63 KO eliminates basal
cells
Gfi-1 KO
reduces
number of
PNECs
Early Saccular phase (E17.5-E18.5)
Hypoplastic versus hyperplastic lungs
• Coupling of redued cell proliferation to
increased cell differentiation
• Some mutants have smaller lungs that
are "immature", other mutants have
larger lungs that are "immature"
• Maturation measured by terminal
differentiation markers in Type II and
Type I epithelial cells.
Saccular phase (E17.5-P5)
Nfib (hyperplastic)
Lung defects in Nfib-/- and +/- E17.5 animals
-/- E17.5 lungs look like E15.5 lungs
suggests maturation defect
Increased DNA content in E18.5
Nfib-/- lungs
# of
Genotype pups
Nfib +/+
6
Nfib -/+
12
Nfib -/5
Total body
weight (g)
1.18  0.065
1.21  0.124
1.38  0.083
Left lung DNA/
Total body wt. (ug/g)
38.6  3.93
51.9  5.08
73.1  10.47
~4X increased PCNA levels in E18.5
Nfib-/- lungs (RT-QPCR)
Loss of differentiation markers
reflect immaturity in Nfib-/- lungs
(RT-QPCR)
E15.5
E17.5
gene
Nfib+/+
Nfib-/Nfib+/+
Nfib-/+
Nfib-/Sftpa
2**
2
100*
40
10
Sftpb
6
3
100
85
39
Sftpc
ND
ND
100
74
48
Shh
146
215
100
ND
214
Vegfa188
20
37
100
39
17
*Signal from E17.5 wild type animals was set to 100% for each gene
and data is reported relative to E17.5 WT controls. (ND, not determined)
Standard deviation 10-15%.
Saccular phase (E17.5-P5)
TGFb3 KO (hypoplastic) E18.5
+/+
-/-
+/+
-/-
Saccular phase (E17.5-P5)
TGFb3 KO (hypoplastic) E18.5
loss of diffn. markers
-/-
Early Saccular phase (E17.5-E18.5)
Hypoplastic versus hyperplastic lungs
• Coupling of reduced cell proliferation to
increased cell differentiation can be
affected differently in different mutants.
• Maturation measured by terminal
differentiation markers in Type II and
Type I epithelial cells. Some mutations
affect expression or proliferation of both
cells types, some only one.
• How many maturation programs are
there and how are they linked?
Saccular phase (E17.5-P5)
Control of epithelial differentiation I
Saccular phase (E17.5-P5)
Control of epithelial differentiation II
This leaves out NFI, GR, Gata6, SP3, Sox11, HIF2A, TGF3
and other TFs and GFs that affect maturation.
Nfib
Alveolarization (P5-P28)
• Terminal differentiation of type I cells
• Further reduction in mesenchyme
• Increased surfactant expression in type
II cells
• Expansion of saccules into alveoli with
septation
Just so you don't think it's too simple
Just so you don't think it's too simple
Just so you don't think it's too simple
Just so you don't think it's too simple
Things we left out
Vasculature
Repair of damage
Lung Stem Cells
Lung diseases
Immune responses
Review
of mouse
lunglung
development
Overview
of mouse
development
Canalicular
Pseudoglandul
ar
Saccular
Alveolar
Cordoso, Dev. Dyn. 219:121 (2000)
Neonatology: SE Wert
5th edition 1999.
Overview
of mouse
development
Review
of mouse
lung lung
development
Canalicular
Pseudoglandul
ar
Saccular
Alveolar
Cordoso, Dev. Dyn. 219:121 (2000)
Neonatology: SE Wert
5th edition 1999.
How to build an organ
Reciprocal Epithelial-Mesenchymal interactions
mediated by growth factor- and cell contactregulated transcriptional networks.
These interactions both constitute a program for
growth, morphogenesis and differentiation and
then help establish the quazi-stable epigenetic
states that characterize each differentiated cell.
Many organs follow this strategy with the addition
of other processes such as cell-recruitment,
Epithelial-Mesenchymal transitions (EMT) and
Mesenchymal-Epithelial transitions (MET).