Normal and abnormal lung development
in mouse and man
Robbert RottierDick Tibboel
Erasmus MC – Sophia Children’s HospitalDepartment of Pediatric Surgery
Rotterdam, The Netherlands
Topics of this presentation
- General introduction early lung development
- Role of Sox2 in lung development
- Hif2α and type II cell differentiation
- Pulmonary hypertension of the newborn
?
Cardoso, 2006
Embryonic origin of the lung
Wells and Melton, 2000
Regionalization of the primitive gut
Serls et al., 2005
Signals emanate from the heart ...
Specification of prospective lung field
Desai et al., 2006
Specification of prospective lung field
Tbx4 induces buds from the gut and local Fgf10Sakiyama et al., 2003
Fgf10 induction !
Tbx4 misexpression
Ectopic bud formation
Lung bud induction
Morrisey and Hogan, 2010
Regionalization of lung bud
Lung development
?
Primitive gut towards complex lung?
alveolar
saccular
canalicular
embryonic
pseudoglandular
growth
differentiation
morphogenesis
embryogenesis
gestational time
Processes during lung development
Branching morphogenesis
Repetitive branching
Early lung development,bronchial tree formation
Branching morphogenesis
drosophila
examples….
kidney
mammary gland
lung
Branching morphogenesis
Lung tissue contains all information to branch
Branching morphogenesis
Fgf-10 Fgf-10 Fgf-10Fgf-10
Fgf-10 induces epithelial branching
Weaver et al., 2000
Branching morphogenesis
Expansion and growth through morphogen gradient
+/+ -/- +/+ -/-
Sekine etal, 1998
lung agenesis
Fgf-10
Branching morphogenesis
Branching morphogenesis
Metzger etal, 2008
3 basic, repetitive processes:
1:
2:
3:
Genes involved in early lung development
Foxa2
Gli2/Gli3Fgf10
Ttf1 Shh
RAR/RAR
HNF4
Gata-6
Foxa2Gli2/Gli3Ttf1; Shh
failure to form foregut endoderm
failure to separate the tracheoesophageal tube
agenesis of lung and trachea
RAR/RAR tracheoesophageal fistula, agenesis of left lung and right lung hypoplasia
gene mutation and major congenital anomalies
foregut
lung bud trachea
lung
esophagus
Lung development
Topics of this presentation
- General introduction early lung development
- Role of Sox2 in lung development
Important for development and cell fate•Sox2 inactivation in mice results in early embryonic lethality•
Sox2 – general
• Sox2 mutations result in congenital anomalies in humans
Sry-box related gene 2, transcription factor•
Sox2 expression during lung development
Sox2 expression is exclusively expressed at the non-branching airways (time and spatial pattern).
E9.5 E10.5 E11.5 E16.5 PN21
I II III IV V
(Dev. Biol. 2008; PhD thesis project SSWO 413; Cristina Gontan-Pardo)
Is downregulation of Sox2 at branching points required for correct lung development?
Key question:
Role of Sox2 in lung development
Maintain Sox2 expression at these branching points
Inducible overexpression of Sox2 in airway epithelium
Role of Sox2 in lung development
rtTASPC
X
Sox2TRE
Inducer Target
Sox2TRErtTASPC Sox2TRE
+ Dox
Inducible overexpression of Sox2+ Dox
rtTA Sox2SPC
Sox2 induces development of progenitors
Gontan et al., Dev Biol 2008
Neuroendocrine cells
Basal cells
CCAM morphology in Sox2 mice?
Comparable cystic lesions??
CCAM type:
proximal distal
affected region:bronchial acinar
clinical prognosis:
poor better
Congenital Cystic Adenomatoid Malformation
Congenital Cystic Adenomatoid Malformation
gestational age
18.59.56.5 14.512.5 16.515.5 17.513.511.510.5
embryonal
pseudoglandular
canalicular
saccular
Dox induction:
embryonal saccular
Experimental CCAM?
Putative CCAM pathology in Sox2 mice?
Gontan et al., Dev Biol 2008
type I
type II
Sox2 staining in human CCAMs
Sox2 DT +DoxSox2 DT -Doxcontrolt
= 0
t =
48 h
rs
In vitro analysis of lung development
Sox2 induced pathology in mice
CCAM ?
control Sox2+
Embryo
control Sox2+
Adult
Emphysema ?
related?
Specific activity of Sox2 is directed by:
Sox2 partners
temporal and spatial expression pattern
associating factors for specificity
Mouse expression profile
Role of Sox2 in the lung
Sox2 in the lung is required for…
control Sox2+
Branching morphogenesis … and cellular differentiation
cont
rol
Sox2
+
cGRP p63
Is this the result of different partners that associate with Sox2?Or, different DNA targets of Sox2? Or both?
Nuclear extracts of ES cells, and Sox2 complex(es) purification using
streptavidin beads
HMG
Sox2Bio-tag
Protein Remarks, (o)= Orkin, Nanog/Oct4 binding
Number peptides
Bio-Sox2Sox2 ESC self-renewal factor 7Oct4 ESC self-renewal factor (o) -Oct1/2/11 Oct1 and Sox2 regulate Hoxb1Nanog ESC self-renewal factorTbx3 ESC self-renewal factor (o) 2Stat3 LIF-dep. self-renewalGATA factor Mesodem differentiation 1sall4 binds Nanog and Oct4 (o)CBF-alpha2 Hematopoetic differentiationFGF receptorCdc2 cell cycle 2HDAC2 NuRD complex 3HDAC3Exportin 4 Nuclear export 2Foxk1 forkhead box K1
Cell differentiation 2
Forkhead box D Neural crest formation p66alpha (GATAd2a)
Transcriptional repressor
Domino Essential for Drosophila somatic stem cell renewal, SNF2 ATPase
TAF9Fetal Alzheimer antigen
= NURF301, H3K4m binding protein, ISWI-partner
BRCA2 DNA-damage protein, breast cancerEMSY Binds BRCA2, breast cancerNAC1 ESC self-renewal factor (o)P300 HAT 2RCC1 Also in Oct4 controlTcf factorZfp219 protein (o)bioSox2
Sox2
control ES bioSox2 ES
I U B I U B
Sox2 partners and targets
bioSox2 ES cells
bioSox2strep
Sox2 and Exportin4Sox2 IgGIP:
a-Xpo4
a-Sox2
WB:
Gontan et al., JCB 2009
Sox2 and human congenital anomalies
N. Ragge et al., AJ of Medical Genetics, 2005
Sox2 linked to severe structural malformations of the eye and AEG syndrome
What is the influence of partners associated with Sox2?
Unexpectedly, the exportin4-Sry interaction explains why certain Sry mutations lead to sex reversal and campomelic dysplasia
Anophthalmia-Esophageal-Genital
Sox2 and human congenital anomalies
Other partner identified: CHD7
CHD7 is linked to CHARGE syndrome, which shares features with AEGColoboma of the eye, Heart defects, Atresia of the nasal choanae, Retardation of growth, Genital abnormalities, Ear abnormalities
Sox2
CHD7
EtBr- + - +CHD7IgG
inp- + - +Sox2IgG
inp
Engelen et al, Nature Genetics, 2011
Sox2 has been associated with syndromes with lung-related abnormalities and tracheal defects
However, its precise role remains uncertain, therefore, elucidating partners may reveal the molecular mechanism(s)
underlying congenital disorders, or unexpected links
Sox2 and human congenital anomalies
Solution(s) for congenital anomalies
Clinicians
“Forward genetics”
Analyze natural occuring “mutants”
(phenotype -> genotype)“Reverse genetics”
Scientists
Modify normal into abnormal situation
(genotype -> phenotype)
Lung developmental group
Dick Tibboel
Cristina Gontan
Yadi HuangJoshua Ochieng
Lalini Raghoebir Ilona Sluiter Marjon Buscop-van Kempen
Anne Boerema-de Munck
Erik Engelen
Raymond Poot’s lab
Ron Smits’ lab
Irwin Reiss
Frank Grosveld
Cell Biology: Pathology:
Ronald de Krijger
Gastroenterology:
Dept. Pediatric Surgery:
Kim Schilders
University of Giessen:
Poornima Mahavadi
Andreas GüntherKatarina Biermann
Genetics:
Siska Driegen
Dies Meijer
Biomics/bioinformatics:
Jeroen Demmers
Sigrid Swagemakers
Topics of this presentation
- General introduction early lung development
- Role of Sox2 in lung development
- Hif2α and type II cell differentiation
- Pulmonary hypertension of the newborn
Congenital Diaphragmatic Hernia
Life-threatening disorder
Pathology
Variable pulmonary hypoplasia
Diaphragm defect
(Surfactant dysfunction)
Pulmonary hypertension
1 of every 2000-3000 live births (high mortality/morbidity)
8% of all major congenital anomalies
Posterolateral Bochdalek (85%), the anterior Morgagni hernia, and the hiatus hernia.
Pulmonary vascular disease
Congenital Diaphragmatic Hernia (CDH):
pulmonary hypertension/vascular disease
lung hypoplasia
Factors are involved in pulmonary hypertension?
ARNT/Hif-1ß
Stable subunit O2 sensitive subunits
Hif1, Hif2, Hif3
Hypoxia dependent gene regulation is mediated by the Hypoxia Inducible transcription Factor-1 (HIF-1)
NAD CADbHLH PAS ODD
transactivation and regulationdimerization and DNA binding
P530 N851
Pulmonary vascular disease
Lee and Percy, 2010
Pulmonary vascular disease
Hif2 is involved in hypoxia-induced pulmonary hypertension
Brusselmans et al., 2003
Pulmonary vascular disease
PH
Control
Hif2α increased in pulmonary hypertension cases
Pulmonary vascular disease
Compernolle et al., 2002
Pulmonary vascular disease
Hif2 in typeII cells
+ Dox
SPC rtTA
Lung-specific
mutHif2
General
Dou
ble
tran
sgen
icCo
ntro
l
air space size (mm2)
Con DT
Pups suffer from respiratory distress with enlarged alveolar structures
Ectopic expression of mutant Hif2
Control Double transgenic
T1a
Sftpc
Ectopic expression of mutant Hif2
Con
DT
Con
DT
Type I Type II
Alveolar epithelial cells
Myc epitope
Cont
rol
Dou
ble
tran
sgen
ic
Toluidine blue
Enlarged cells, more surfactant production ??
Ectopic expression of mutant Hif2
mutHif2 expressing cells accumulate glycogen(a precursor for phospholipids)
PAS PAS + amylase -Myc
Ectopic expression of mutant Hif2
Ectopic expression of mutant Hif2
Hif2a expression leads to down regulation of Lpcat1 and Abca3
Lpca
t1A
bca3
Double transgenicControl
Ectopic expression of mutant Hif2
mutHif2 causes changes in phospho-lipid composition
respiratory distress
glycogen accumulation in epithelial cells
abnormal type II cells differentiation
Oxygen-insensitive Hif2 expression results in:
Ectopic expression of mutant Hif2
Topics of this presentation
- General introduction early lung development
- Role of Sox2 in lung development
- Hif2α and type II cell differentiation
- Pulmonary hypertension of the newborn
Pulmonary hypertension is a serious complication, requiring intensive care:
Reduced or lack of gas-exchange in the lung
Pulmonary hypertension leads to hypoxemia in patients
Pulmonary hypertension eventually leads to changes in the vascular wall (media – VSMC)
- Increased vessel wall thickness, neomuscularization- Abnormal vasoreactivity- Disorganized postnatal vascular remodelling
Pulmonary hypertension
• Structural changes:
- persistent or augmented thickening of the vessel wall
- extensive muscularization of micro vessels (neo-muscularization)
Michelakis et al, 2008
Pulmonary hypertension of the newborn
migration +proliferation increased ECM production contractility
Maturation of smooth muscle cells
Pulmonary hypertension
cell size ↑extracellular matrix production ↑contractile protein expression ↓
migration ↑
cell size ↓extracellular matrix production ↓contractile protein expression ↑
migration ↓
Rensen et al.,2007:
VSMCs may have different phenotypes:
CRBP-1SM MHCSmoothelin
Pulmonary hypertension
Proximal-distal gradient in lung maturation
Gradual maturation of pulmonary vessels (and airways) along the proximo-distal axis
Parera et al, 2005
Ergun, 2010Hislop, 2005
proximal
distal
Smooth muscle cells
Pericytes
Established nitrofen rat model for CDH
Hypoplastic left lung
Herniated liver
Used to study CDH and pulmonary hypertension of the newborn
HeLu
Lu Li
Human lung (infant)
CDH
cont
rol
α-SMA Calponin SMMHC
Pulmonary hypertension
α-SMA
SMMHC 1
γ-SMA
Calponin
Caldesmon
SMMHC 2
SMC differentiation
prematureα-SMA SM-MHC Calponin Myl-9 CRBP-1
CDHcontrol
proximal
distal
Hislop A, Ped Resp Rev, 2005
Pulmonary hypertension
Pulmonary hypertension
Proximo-distal gradient of differentiated VSMCs
Parera M, 2005
Vascular growth by (distal) angiogenesis:
Donate, 2005
Potente, 2011
Pericyte recruitment stabilizes primitive sprouts
Pulmonary hypertension
E 17 E19 E 21
PDGF
rβ/ α-
SMA
NG2
/ α
-SM
ACharacterization of pericytes in normal fetal rat lung:
Pulmonary hypertension
Rat lung tissue
control CDH
200x
1000x
-SMANG2
Pulmonary hypertension
The role of the smooth muscle cells in pulmonary hypertension?
Distinct vascular differences
Structural differences in PA-SMCs
Functional differences in PA-SMCs
Peri-vascular cells / Pericytes: SMC precursors?
controlCDH
Pulmonary hypertension
Solution(s) for congenital anomalies
Clinicians
“Forward genetics”
Analyze natural occuring “mutants”
(phenotype -> genotype)“Reverse genetics”
Scientists
Modify normal into abnormal situation
(genotype -> phenotype)
Lung developmental group
Dick Tibboel
Cristina Gontan
Yadi HuangJoshua Ochieng
Lalini Raghoebir Ilona Sluiter Marjon Buscop-van Kempen
Anne Boerema-de Munck
Erik Engelen
Raymond Poot’s lab
Ron Smits’ lab
Irwin Reiss
Frank Grosveld
Cell Biology: Pathology:
Ronald de Krijger
Gastroenterology:
Dept. Pediatric Surgery:
Kim Schilders
University of Giessen:
Poornima Mahavadi
Andreas GüntherKatarina Biermann
Genetics:
Siska Driegen
Dies Meijer
Biomics/bioinformatics:
Jeroen Demmers
Sigrid Swagemakers
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