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CONGENITAL DIAPHRAGMATIC HERNIA

JOHN J. GREERUNIVERSITY OF ALBERTA

MAIN TOPICS

1. ORIGIN OF DIAPHRAGM DEFECT

2. RETINOID SIGNALLING AND GENES LINKED TO CDH

ANIMAL MODELS OF DIAPHRAGMATIC HERNIA

TERATOGENIC

DIETARY (VITAMIN A DEFICIENT)

GENETIC

CONTROL NITROFEN VITAMIN A DEFICIENT

Diaphragm defects in all rodent models are consistently located in the postero-lateral corner of the diaphragm, in parallel with human cases of Bochdalek CDH

WT1 -/- COUP-TFII

- * muscle limited to smallregion

- eventration-like hernia- independent lung hypoplasia

Fog-2 MUTANT DIAPHRAGM(Kate Ackerman)

- Herniation in septumtransversum

Liver

SEPTUM DEFECT(GATA4/Slit3)

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QUESTION: HOW DOES THE DIAPRHAGM FORM IN UTERO?

QUESTION: WHAT ASPECT OF THE DEVELOPMENTIS ABNORMAL TO EXPLAIN THE DIAPHRAGM DEFECT ASSOCIATED WITH CDH?

E14.5 E15.5

E16.5

Centraltendon Central

tendon

Location of the primordialdiaphragm

ONTOGENY OF DIAPHRAGM MUSCULARIZATION

E17.5

Centraltendon

Centraltendon

Week 10 inHuman –onset of fetal breathing movements

PRIMORDIAL TARGET FOR DIAPHRAGM MUSCLE PRECURSORSAND PHRENIC AXONS – PLEUROPERITONEAL FOLD (PPF)

MyoD

H

Lu

SC

Pax3PPF Location

Phrenic nerveMuscle precursors

E12.5 E13.0 E13.5 E14

FORMATION OF PPF IN RAT EMBRYOS

Green = muscle precursors (myoD) Red = phrenic axons (NF)

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Primordialdiaphragmmusculature –Pleuroperitonealfold

Septumtransversum

A STRUCTURAL PPF DEFECT IS COMMON IN ALL CDH MODELS

Nitrofen Vitamin A deficient WT1 -/-

Control

3-D RECONSTRUCTION SHOWING COMMON PPF DEFECTS INMULTIPLE RODENT MODELS OF CDH

Building a pathogenic model of CDH

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QUESTION:WHY IS THE DIAPHRAGM THE ONLY MUSCLEAFFECTED IN CDH – INCLUDING ANIMAL MODELS?

defect

HYPOTHESIS:IT IS NOT A PROBLEM RELATED TO MUSCLE FORMATION PER SE

PROPOSED ANSWER:IT IS A PROBLEM WITH THE NON-MUSCULAR COMPONENT OF THE PRIMORDIAL DIAPHRAGM (I.E. MESENCHYMAL TISSUE)

NITROFEN-INDUCED CDH INc-met NULL-MUTANT MICE- THESE MICE HAVE AN AMUSCULAR DIAPHRAGM

c-met-/-

amusculardiaphragm

liver defect

HYPOTHESIZED VIEW OF DEFECT IN THE MUSCULARIZATION OF THE PPF

Muscle adjacent to the defect is thicker

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QUESTIONS:

IS THERE A PPF IN THE HUMAN EMBRYO?

WHAT STAGE DOES IT DEVELOP (INSIGHTS INTO THE INITIAL TIMING OF CDH INSULT IN HUMANS)?

DOES THE APPEARANCE OF THE HUMAN DIAPHRAGMIN CDH CORRELATE WITH RODENT DATA (I.E. THICKENING OF TISSUE AROUND THE HOLE)?

HUMAN PPF SIMILAR TO RODENT

Carnegie stage 13-17; gestational week 4-6

Human Rat

TIMELINE OF PPF FORMATION: HUMAN VS RAT

nitrofenPPF

defect

E0 E20E12-E14

RAT GESTATION

HUMAN GESTATION

Wk 0 Wk 38Wk 4-6

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defect

QUESTIONS:ARE THE GENES LINKED TO CDH EXPRESSED WITHIN THE DEVELOPING PPF?

IF SO, ARE THEY EXPRESSED IN THE SAME CELL-TYPE?

IF SO, ARE THEY EXPRESSED IN THE NON-MUSCULAR MESENCHYMAL CELLS AS PREDICTED BY THE MODEL OF CDH PATHOGENESIS?

Methods 1: mRNA expression studies

RN

A E

XT

RA

CT

ION

RT-P

CR

Whole Embryo (E13.5)

PPF (E13.5)

Whole Diaphragm (E16.5)

Wt1

Coup-tfII

Gata4

Fog2

Results 1: mRNA expression studies

All CDH-related transcription factors are expressed in the PPF and the fully formed diaphragm.

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Results 2: Protein expression studiesWT1, COUP-TFII, GATA4 and Fog2 (GREEN) are expressed through-out the PPF, but do not co-localise with Pax3 positive muscle precursor cells (RED).

Results 3: Protein expression studies

Wt1, Coup-tfII, Gata4 and Fog2 are all co-expressed in the PPF

1. The pleuroperitoneal fold (PPF) is the major, if not sole, source of diaphragmatic musculature

2. A defect in the mesenchymal substratum of the PPF underlies the pathogenesis of nitrofen-induced CDH

SUMMARY: ORIGINS OF DIAPHRAGM AND SOURCEOF CDH DEFECT

SUMMARY: CDH GENE EXPRESSION IN PPF

1. All are expressed in the PPF and the fully formed diaphragm

2. All are co-expressed within the same cells of the PPF

3. But, do not co-localize with muscle precursor cells in the PPF (i.e. support mesenchyme hypothesis)

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HYPOTHESIS:Perturbation of retinoid signalling can result in CDH

1. Vit A deficient rats have diaphragmatic hernias similar to CDH (reports in 1940-50s).

RATIONALE:

2. Some offspring of null-mutants missing multiple retinoic acid receptor subtypes have diaphragmatic defects (Mendelsohn, 1994)

3. Retinoid signalling interacts with genes implicated in CDH

4. Teratogens that induce CDH in rodents decrease retinoic acid synthesis

Control (C)

Nitrofen-exposed (N)

Nitrofen + Retinoic acid(N+RA)

RETINOID RESPONSE ELEMENT ACTIVATION SUPPRESSED BY NITROFEN

Retinol-CRBPRetinolRetinal

DehydrogenaseDehydrogenase

pre-mRNA

RARE PROMOTER-LacZ GENEDNA

Retinoic acidRetinoic acid

Nucleus

Cytoplasm

Retinyl esters Retinol Retinol-RBP

Retinol-RBP QUESTION: WHICH ASPECTS OF THE RETINOID SIGNALLING PATHWAY ARE SPECIFICALLY EXPRESSED WITHIN THE AMUSCULAR MESENCHYME OF THE PPF?

YES

RALDH2CrabpIIRXRαRARαRARγ

NO

RALDH1, RALD3CrabpIRXRβRXRγRARβ

QUESTION: ARE ANY OF THE RETINOIC ACID PATHWAYMEMBERS EXPRESSED IN THE SPECIFIC REGION OF THE PPF THAT IS MALFORMED IN CDH MODELS?

RARα

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RARα expression domain reflects lost tissue in animal models of CDH

RARα Nitrofen exposed

rostral

caudal

PREDICTION:BLOCK OF RARαWITH A RECEPTOR ANTOGONIST(BMS493) WILL INDUCE BOCHDALEK CDH

DRUG SYNTHESIZED BY Angel R. de LeraProfessor of Organic ChemistryUniversidade de VigoVigo, Spain

AGE-DEPENDENT SUSCEPTIBILITY TO CDH BETWEEN LEFT AND RIGHT SIDE

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MAJOR UNRESOLVED ISSUES

EMBRYOGENESIS OF THE PPF AND CELL COMPOSITION?

WHICH ASPECTS OF PPF EMBRYOGENESIS IS AFFECTEDIN CDH (I.E. CELL ADHESION/PROLIFERATION, APOPTOSIS)?

WHAT IS THE INTERACTION BETWEEN ‘CDH GENES’AND RETINOID SIGNALLING?

HOW DO THOSE GENES AND RETINOIDS CONTROLPPF FORMATION?

ARE THERE DEFICIENCIES IN RETINOID LEVELS ORRELATED PROTEINS IN UTERO ASSOCIATED WITH CDH?

FUNDING: CIHR, AHFMR, and March of Dimes

COLLABORATORS

DR MARGARET CLAGETT-DAME University of WisconsinDR. CHRISTOFF ENGLERT University of JenaDR. JALENA MARTINOVIC Hôpital Necker-Enfants MaladesDR. ALEXNDRA BENACHI Hôpital Necker-Enfants MaladesDR. JORGE MEY Institute of Biology, Aachen DR. CLAES BAVIK University of Texas at AustinDR. KATE ACKERMAN University of RochesterDR. RUNE BLOMHOFF University of OsloDR. PAUL WALFISH University of TorontoDR. DAVID ORNITZ Washington UniversityDR. BERNARD THEBAUD University of AlbertaDR. DICK TIBBOEL Erasmus University

GRADUATE STUDENTS RESEARH ASSOCIATE

DOUGLAS ALLAN: 1994-1999 WEI ZHANG: 1996-present

RANDAL BABIUK: 1998-2003

RHIANNON NOBLE: 2003-2005

ROBIN CLUGSTON: 2003-2008

DARINE ROUM: 2008-present

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Nitr

ofen

VA

D8

VA

D10

VA

D8,

10

VA

D8,

10,1

2

RA

D8

RA

D8-

10

RA

D8-

13

0.00

0.25

0.50

0.75

Her

nia

Inci

denc

e(h

erni

ated

/tot

al#)

* * *

** *

53%

32%

15%

Nitrofen +Vit A

Nitrofen + retinoic acid

Nitrofen

RALDH2Retinoic acid RetinalX

HYPOTHESIS:ADMINISTRATION OF RETINOIDS WITH NITROFEN WILL DECREASE THE INCIDENCE OF HERNIAS