Normal and abnormal lung development in mouse and man · Normal and abnormal lung development in...

Normal and abnormal lung development

in mouse and man

Robbert RottierDick Tibboel

[email protected]

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


drosophila

examples….

kidney

mammary gland

lung


Lung tissue contains all information to branch


Fgf-10 Fgf-10 Fgf-10Fgf-10

Fgf-10 induces epithelial branching

Weaver et al., 2000


Expansion and growth through morphogen gradient

+/+ -/- +/+ -/-

Sekine etal, 1998

lung agenesis

Fgf-10



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

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


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


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

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


Lee and Percy, 2010


Hif2 is involved in hypoxia-induced pulmonary hypertension

Brusselmans et al., 2003


PH

Control

Hif2α increased in pulmonary hypertension cases


Compernolle et al., 2002


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


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 ??


mutHif2 expressing cells accumulate glycogen(a precursor for phospholipids)

PAS PAS + amylase -Myc



Hif2a expression leads to down regulation of Lpcat1 and Abca3

Lpca

t1A

bca3

Double transgenicControl


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:


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


• 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


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


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


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



Proximo-distal gradient of differentiated VSMCs

Parera M, 2005

Vascular growth by (distal) angiogenesis:

Donate, 2005

Potente, 2011

Pericyte recruitment stabilizes primitive sprouts


E 17 E19 E 21

PDGF

rβ/ α-

SMA

NG2

/ α

-SM

ACharacterization of pericytes in normal fetal rat lung:


Rat lung tissue

control CDH

200x

1000x

-SMANG2


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


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

Normal and abnormal lung development in mouse and man · Normal and abnormal lung development in...

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