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  • Normal and abnormal lung development

    in mouse and man

    Robbert Rottier Dick Tibboel

    r.rottier@erasmusmc.nl

    Erasmus MC – Sophia Children’s Hospital Department 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 Fgf10 Sakiyama 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

    Foxa2 Gli2/Gli3 Ttf1; 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 Sox2 TRE

    + 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 -Doxcontrol t

    = 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

    co nt

    ro l

    So x2

    +

    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-Sox2 Sox2 ESC self-renewal factor 7 Oct4 ESC self-renewal factor (o) - Oct1/2/11 Oct1 and Sox2 regulate Hoxb1 Nanog ESC self-renewal factor Tbx3 ESC self-renewal factor (o) 2 Stat3 LIF-dep. self-renewal GATA factor Mesodem differentiation 1 sall4 binds Nanog and Oct4 (o) CBF-alpha2 Hematopoetic differentiation FGF receptor Cdc2 cell cycle 2 HDAC2 NuRD complex 3 HDAC3 Exportin 4 Nuclear export 2 Foxk1 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

    TAF9 Fetal Alzheimer antigen

    = NURF301, H3K4m binding protein, ISWI-partner

    BRCA2 DNA-damage protein, breast cancer EMSY Binds BRCA2, breast cancer NAC1 ESC self-renewal factor (o) P300 HAT 2 RCC1 Also in Oct4 control Tcf factor Zfp219 protein (o)bioSox2

    Sox2

    control ES bioSox2 ES

    I U B I U B

    Sox2 partners and targets

    bioSox2 ES cells

    bioSox2 strep

  • Sox2 and Exportin4 Sox2 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 AEG Coloboma 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 Günther Katarina Biermann

    Genetics:

    Siska Driegen

    Dies Meijer

    Biomics/bioinformatics:

    Jeroen Demmers

    Sigrid Swag