J Appl Physiol
89: 2123–2129, 2000.
Structure of the primordial diaphragm and defects
associated with nitrofen-induced CDH
JOHN J. GREER,1 DAVID COTE,2 DOUGLAS W. ALLAN,1 WEI ZHANG,1
RANDAL P. BABIUK,1 LINH LY,1 ROBERT P. LEMKE,3 AND KEITH BAGNALL2
Departments of 1Physiology, 2Anatomy, and 3Pediatrics, University of Alberta,
Edmonton, Alberta, Canada T6G 2S2
Received 8 June 2000; accepted in final form 5 July 2000
have identified the pleuroperitoneal fold (PPF) as a primary source of diaphragmatic musculature (1). The PPF is a pyramidshaped tissue that extends medially from the lateral
cervical wall to the esophageal mesentery and fuses
ventrally with the septum transversum. The early
stages of PPF formation have yet to be systematically
examined. However, we hypothesize that the embryogenesis of the PPF parallels that of the forelimb bud.
Specifically, the substructure of the PPF is derived
from mesenchymal cells migrating from the somatic
mesoderm. Subsequently, muscle precursors migrating
from the dermomyotome of cervical somites follow
guidance cues provided by the somatopleure substruc-
ture and become localized in the PPF (www.med.
unc.edu/embryo_images provides a clear pictorial review of early embryological events). The PPF becomes
a well-recognized structure between embryonic days
(E) 12.5 and 13.5 in the rat (gestational period is 21
days). The key development stages of phrenic nerve
and diaphragm development after the formation of the
PPF have already been elucidated (see Ref. 8 for review). Briefly, the phrenic nerve makes contact with
the PPF during E13. Between E14 and E17, the
phrenic nerve trifurcates and extends intramuscular
branches to the ventral, dorsolateral, and crural areas
of the developing diaphragm. Concomitant with
phrenic branch outgrowth, diaphragmatic myoblasts
migrate from the PPF and fuse to form myotubes that
extend from the lateral aspect of the septum transversum to the body wall in the costal areas and from the
esophageal mesentery to the dorsal aspects of the crural region. The tissue derived from the septum transversum eventually becomes surrounded by diaphragmatic muscle fibers and forms the central tendon.
A clear understanding of PPF structure is fundamental for an understanding of the early stages of
diaphragm embryogenesis. Furthermore, the PPF has
become a focus of attention in studies of the pathogenesis of an animal model of congenital diaphragmatic
hernia (CDH) (2). This often-lethal developmental
anomaly (⬃50% mortality) is estimated to occur in
1:3,000 births (6, 9, 16). The hallmark of the disorder is
a hole in the dorsolateral region of the diaphragmatic
musculature [with 85–90% of the defects occurring on
the left side (16)]. The presence of a hole in the diaphragm allows for the invasion of the thoracic cavity by
the growing abdominal contents and the subsequent
impairment of normal lung growth. There is a wellestablished animal model of CDH in which defects very
similar to those observed in the human condition are
produced in rodent embryos by administering a single
dose of the herbicide nitrofen to the dam on E8–9 (3, 5,
15). The biochemical mechanism by which nitrofen
produces the defects is, at present, unknown. Nevertheless, the model has proven useful for testing several
Address for reprint requests and other correspondence: J. J. Greer,
Dept. of Physiology, Div. of Neuroscience, 513 HMRC, Univ. of
Alberta, Edmonton, AB, Canada T6G 2S2 (E-mail: John.Greer
@Ualberta.ca).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
embryology; myogenesis; lung hypoplasia; pleuroperitoneal
fold; congenital diaphragmatic hernia
RECENT EMBRYOLOGICAL STUDIES
http://www.jap.org
8750-7587/00 $5.00 Copyright © 2000 the American Physiological Society
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Greer, John J., David Cote, Douglas W. Allan, Wei
Zhang, Randal P. Babiuk, Linh Ly, Robert P. Lemke,
and Keith Bagnall. Structure of the primordial diaphragm
and defects associated with nitrofen-induced CDH. J Appl
Physiol 89: 2123–2129, 2000.—The goals of this study were
to further our understanding of diaphragm embryogenesis
and the pathogenesis of congenital diaphragmatic hernia
(CDH). Past work suggests that the pleuroperitoneal fold
(PPF) is the primary source of diaphragmatic musculature.
Furthermore, defects associated with an animal model of
CDH can be traced back to the formation of the PPF. This
study was designed to elucidate the anatomic structure of the
PPF and to determine which regions of the PPF malform in
the well-established nitrofen model of CDH. This was
achieved by producing three-dimensional renderings constructed from serial transverse sections of control and nitrofen-exposed rats at embryonic day 13.5. Renderings of leftand right-sided defects demonstrated that the malformations
were always limited to the dorsolateral portions of the caudal
regions of the PPF. These data provide an explanation of why
the holes in diaphragmatic musculature associated with
CDH are characteristically located in dorsolateral regions.
Moreover, these data provide further evidence against the
widely stated hypothesis that a failure of pleuroperitoneal
canal closure underlies the pathogenesis of nitrofen-induced
CDH.
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PRIMORDIAL DIAPHRAGM DEFECTS IN CDH
hypotheses regarding the stage at which the initial
malformation of the diaphragm occurs (see Ref. 7 for
review). Studies using the nitrofen model have demonstrated that the initial defect can be traced back to the
formation of the PPF.
The present study focuses on two main objectives: 1)
to elucidate the anatomic structure of the primordial
diaphragm and 2) to determine which regions of the
PPF malform in the animal model of CDH and how
these early defects could translate into the characteristic holes in the dorsolateral quadrant of the defective
newborn diaphragm. These aims were achieved by
generating three-dimensional (3D) reconstructions of
normal and defective PPFs from serial transverse sections of control and nitrofen-exposed rat embryos. The
reconstructions were generated from tissue isolated at
E13.5, when the PPF has formed into a well-defined
structure and defects are clearly discernible in the
nitrofen model (1, 2).
Administration of nitrofen. Nitrofen was obtained from the
US Environmental Protection Agency and prepared as a
solution of 100 mg/ml in olive oil. The timing of pregnancies
was determined from the appearance of sperm plugs in the
breeding cages (designated as E0). Pregnant dams on the
evening of the eighth day of gestation (E8) were temporarily
(⬃10 min) anesthetized with halothane (1.25% in 95% O2-5%
CO2) and given 100 mg of nitrofen via a gavage tube. Past
work has demonstrated that nitrofen produces diaphragmatic defects in 30–90% of the embryos in a given dam, with
the majority being left-sided defects (2, 15).
Delivery of embryos via caesarean section and tissue preparation. Control and nitrofen-exposed fetal rats (E13.5) were
delivered from timed-pregnant Sprague-Dawley rats anesthetized with halothane (1.25% in 95% O2-5% CO2), as approved by the Animal Welfare Committee at the University
of Alberta. The ages of fetuses were confirmed by comparison
of crown-to-rump length measurements with those published
by Angulo y González (4). This measurement is crucial for
determining the precise age of fetuses. Embryos were decapitated, and the thoracic and abdominal cavities were exposed
while immersed in 4% paraformaldehyde in a 0.2 M sodium
phosphate buffer (pH 7.4). The tissue was postfixed at 4°C for
4–20 h, dehydrated in a graded series of ethanols, and
cleared in xylene. The fixed tissue was embedded in molten
paraffin in a heating incubator at 54°C for 2–3 days. After
tissues were embedded, the paraffin block containing the
tissue was trimmed and mounted on a rotary microtome
(model 45, Lipshaw, Detroit, MI) equipped with a standard
knife holder and a forward-moving block holder. The tissue
was orientated to allow for the cutting of transverse sections
(6 ␮m thickness). Sections were mounted on gelatin-subbed
microscope slides and immersed in xylene for 15 min to
remove wax before staining. The dewaxed sections were
dehydrated through a graded series of ethanols, stained with
Gill’s hematoxylin for 2 min for nuclear staining, and rinsed
with distilled water. The sections were then stained for 1 min
with eosin for cytoplasmic staining [collectively referred to as
hematoxylin and eosin (H & E staining)]. After they were
washed with distilled water, the sections were dehydrated in
ethanol, equilibrated in xylene, and positioned under a glass
cover with Entellan.
RESULTS
Two-dimensional PPF structure. The location of the
PPF within a cervical section of an E13.5 rat is illustrated in Fig. 1A. PPF tissue is clearly discernable from
surrounding structures (e.g., lung, liver, and body
wall), on the basis of differential cell density and size
revealed by H & E staining. The adjacent photomicrograph (Fig. 1B) shows a close-up image of the PPF
tissue labeled for the low-affinity nerve growth factor
(p75) receptor, one of several molecules (e.g., neural
cell adhesion molecule, desmin) that labels the primordial diaphragmatic muscle mass (1, 2). We used the
combination of differential H & E staining and immunolabeling profiles of developmentally regulated molecules, from previous studies, to establish the boundaries for delineating the PPF. The rostral border was
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METHODS
Immunolabeling for the low-affinity nerve growth factor
(p75) receptor and polysialylated neural cell adhesion molecule was performed as previously described (1, 2).
Image processing and 3D reconstructions. The thoracic
region of each embryo was sectioned serially. The sections
were then screened for obvious defects within the PPF. Subsequently, serial transverse sections from representative embryos (with and without PPF defects) were chosen for 3D
reconstructions. To meet the criteria for reconstruction, the
series of tissue sections from a given embryo had to be
structurally complete throughout the rostrocaudal extent of
the PPF. Photomicrographs of each transverse section were
then captured in digital format using Image Pro software
connected to a MCI 3 charge-coupled-device camera mounted
on a Leitz Diaplan microscope (⫻10 objective). Digital images
were imported into CorelDRAW 8.0, in which the drawing
tool was used to outline the PPF. Images were transformed
into gray-scale images and stored as TIFF files. During our
initial reconstructions, each 6-␮m slice spanning the rostrocaudal extent of the PPF was captured and used for the 3D
reconstructions (36–41 sections/PPF). However, with later
reconstructions, we found that capturing every second section sufficed for generating renderings that faithfully depicted PPF structure (17–21 sections/PPF).
The TIFF-formatted images were transferred to Adobe
Photoshop 4.0 for preliminary cropping and for alignment of
the sections, using the esophagus as a fiducial point. The
esophagus was a prominent feature in each of the transverse
sections and appeared in a consistent locale throughout the
limited rostrocaudal extent of the PPF (i.e., ⬍250-␮m thickness). The image sets were opened up as a series in the 3D
rendering program SURFdriver 3.5 (see www.surfdriver
.com). Data for the scaling and dimensions of the tissue
sections were entered into the software program. A calibrated micrometer incorporated into the microscope eyepiece
was used to measure values in the x- and y-dimensions. The
values in the z-direction were derived from the settings on
the cutting microtome. The final fine adjustments necessary
to align the sections were performed in SURFdriver before
the PPF contours were outlined for the rendering of the 3D
image. Once generated, the 3D images were rotated in the
rendering window and captured at the x-, y-, and z-coordinates that we considered optimum for visualizing the overall
shape of the PPF and the regions of nitrofen-induced defects.
Perspective views of the PPF, from both the right and left
sides of the embryos, were selected. The images were then
exported from SURFdriver as “bitmap” files to CorelDRAW
software for the construction and labeling of final figures.
PRIMORDIAL DIAPHRAGM DEFECTS IN CDH
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Fig. 1. Photomicrographs from embryonic day (E) 13.5 rat embryos showing
the location and 2-dimensional shape of
the pleuroperitoneal fold (PPF). A:
transverse section showing the location of the PPF (*) with respect to the
surrounding tissue (hematoxylin and
eosine staining). B: higher magnification of the PPF region (*) from another
E13.5 embryo, in which immunohistochemical labeling for the low-affinity
nerve growth factor (p75) receptor has
labeled the phrenic nerve and primordial muscle tissue within the boundaries of the PPF. SC, spinal cord; H,
heart; Fl, forelimb; Lu, lung; E, esophagus; A, aorta; VC, vena cava.
boundary was estimated to be the point at which the
PPF was no longer clearly discernable from the underlying liver tissue. Note that the liver extends to underlie both the left and right PPFs at this stage of embryonic development (Fig. 2). The extents of the
ventrodorsal and mediolateral boundaries of the PPF
used for the 3D reconstructions are outlined in Fig. 2.
On the basis of the defined boundaries chosen, the
average rostrocaudal extents of normal PPFs were
Fig. 2. A series of transverse sections
taken from the rostral, middle, and
caudal regions of the PPF (*), from 3
different E13.5 embryos. Right: tissue
from a nitrofen-exposed embryo with a
left-sided PPF defect. Middle: normal
tissue from a control animal. Left: transverse sections from an embryo with a
nitrofen-induced right-sided PPF defect. The top-middle image shows an
example of the outline drawn to define
the extent of PPF boundaries for the
purpose of generating 3-dimensional
(3D) reconstructions. Arrows point to
liver tissue that occupies regions in
which the PPF is missing in the nitrofen-exposed embryos. R, right side; L,
left side.
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estimated to be the first serial transverse section in
which the triangle-shaped PPF tissue could clearly be
seen protruding medially from the cervical lateral wall.
The phrenic nerve could be distinguished from the
surrounding tissue within the medial aspect of rostrally located sections of the PPF. The PPF thickens
and extends caudally while maintaining close contact
laterally with the body wall and medially with the
primary esophageal mesentery. The limit of the caudal
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PRIMORDIAL DIAPHRAGM DEFECTS IN CDH
Fig. 3. Representative 3D reconstructions of
a normal PPF and PPFs with left-sided defects. Renderings are shown from the perspectives of looking through the right and left
lateral cervical walls of E13.5 rat embryos. A:
normal left and right PPFs. B–D: 3D reconstructions of left-sided defects of varying degrees (arrows point to defective regions). In
all cases, the defects are in the dorsolaterally
located regions of the caudal PPF. R, right
PPF; L, left PPF. Scale bars ⫽ 100 ␮m.
PPFs. The 3D reconstructions are oriented to provide
views from both the left and right lateral walls. This
allows for a clear visualization of the medial and lateral perspectives of the bilateral PPFs. A comparison of
the left control PPF (Fig. 3A) and the defective PPFs
(Figs. 3, B-D) illustrates that the malformed regions
are always located caudally and dorsolaterally. In contrast, the rostral, ventral, and most medial regions of
the defective and normal PPFs are similar. Furthermore, PPF defects of varying magnitudes, ranging
from mild (Fig. 3B) to moderate (Fig. 3C) to severe (Fig.
3D), were detected.
Figure 4 shows a rendering of a normal PPF and a
PPF with a right-sided defect. As was the case for
left-sided defects, the malformed PPF regions are those
located caudally and dorsolaterally. Only the right PPF
is shown from this embryo because the corresponding
left PPF tissue sections were not of suitable quality for
reconstruction.
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206 ⫾ 23 (SD) and 229 ⫾ 16 ␮m (n ⫽ 18 for both) for
the left and right sides, respectively. All dimensions
are derived from fixed tissue and thus may be underestimated by ⬃15% due to tissue shrinkage.
Figure 2 illustrates cross sections of the PPF at
E13.5 in embryos with normal and malformed PPFs.
Sections are shown from the rostral, middle, and caudal regions of the PPF. The nitrofen-induced defects of
the PPF, whether on the right or left side, were always
found in sections taken from the middle and caudal
regions of the PPF. The single right-sided defect measured was first noticeable at 110 ␮m from the rostral
border of the PPF. The left-sided defects started at an
average of 143 ⫾ 24 ␮m (n ⫽ 11) from the rostral
border of the PPF. Furthermore, the areas of PPF
missing in the caudal sections were consistently located dorsolaterally. The extent to which the defect
encompassed more medial areas of the PPF varied.
3D PPF structure. Figure 3 shows 3D reconstructions
from embryos with and without defective left-sided
PRIMORDIAL DIAPHRAGM DEFECTS IN CDH
2127
Correlation between PPF and diaphragm muscle defects. Figure 5 was constructed to assist the reader in
visualizing how the regional defects of the PPF could
translate into the lack of dorsolateral diaphragmatic
musculature at later stages of development. The defect
in the PPF at E13.5 (Fig. 5A) is located in the typical
dorsolateral portion. A representative, moderate-sized,
nitrofen-induced defect of diaphragm musculature at
E15.5 is shown in Fig. 5B. Note that the area of
diaphragm musculature missing is also located in the
dorsolateral portions. Therefore, although we do not,
as of yet, have a complete mechanistic understanding
of how muscle precursors in the PPF contribute to the
various regions of the diaphragm musculature, the
anatomic profiles of regional defects in the PPF and
diaphragm correlate well.
Fig. 5. Juxtaposed photomicrographs of a defective PPF (E13.5; A) and diaphragm (E15.5; B) illustrating the
correlation between regional defects at the 2 stages of development. A: close-up of the PPF (*) in a transverse
section from a nitrofen-exposed E13.5 embryo (without underlying liver tissue). There is a pronounced malformation (arrow) of the left PPF in the characteristic dorsolateral region. B: diaphragm from an E15.5 embryo with a
classical nitrofen-induced, left-sided defect (arrow). The tissue has been immunolabeled for polysialylated neural
cell adhesion molecule, which is expressed by the phrenic nerve and primary myotubes. Comparing the two images
shows that the defect in the dorsolateral PPF could translate into a hole in the dorsolateral region of the diaphragm
musculature at later stages of development. Furthermore, the size of the defect in the diaphragm musculature
could be dictated by the magnitude of the initial PPF malformation.
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Fig. 4. Representative 3D reconstructions of a normal PPF and a PPF with a right-sided defect. Renderings are
shown from the perspectives of looking through the right and left lateral cervical walls of E13.5 rat embryos. A:
normal bilateral PPF from a control animal not exposed to nitrofen. B: 3D reconstruction of a right-sided defect
(arrows point to defective regions). As was the case for left-sided defects (Fig. 3), the malformation is located in the
dorsolaterally located regions of the caudal PPF. Scale bars ⫽ 100 ␮m.
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PRIMORDIAL DIAPHRAGM DEFECTS IN CDH
DISCUSSION
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The 3D images provide the first clear visualization of
PPF morphology and dimensions in an embryo. Furthermore, the images of nitrofen-induced PPF defects
allow for an appreciation of the precise areas that are
malformed. The realization that the dorsolateral region is the defective area in all cases provides a plausible explanation of why the holes in diaphragmatic
muscle are consistently found in the corresponding
dorsolateral regions. Moreover, these data provide further support against the widely stated hypothesis that
a failure of pleuroperitoneal canal closure underlies
the pathogenesis of nitrofen-induced CDH.
Structure of the primordial diaphragm and PPF.
Textbooks typically provide a brief description of diaphragm embryogenesis that states that the musculature is derived from multiple sources, including the
esophageal mesentry, the septum transversum, and
the body wall musculature (16). However, those descriptions are largely based on speculations derived
from examining the arrangement of muscle fibers in
fully formed embryonic diaphragms (14, 18). Although
issues pertaining to the formation of the diaphragm
remain unresolved, recent data strongly suggest that
the PPF is a major component of the primordial diaphragmatic musculature (1). The septum transversum
is a distinct structure that fuses with the PPF and
forms the central tendon of the diaphragm. The 3D
reconstructions provided in this study allow for a clear
visualization of PPF morphology before the onset of
diaphragm myotube formation. It could be argued that
the rendered 3D image of the PPF would differ if the
boundaries chosen in the tissue sections were varied.
However, unless the boundaries were chosen well outside those suggested by the staining profiles described,
the general shape of the PPF and the identification of
the defective regions in the nitrofen model would not
differ significantly from that presented.
Defects of the PPF associated with the nitrofen model
of CDH. The first sign of a nitrofen-induced defect in
diaphragm tissue can be traced back to the PPF. Studies by Iritani (10) and Kluth et al. (11–13) demonstrated nitrofen-induced defects in the region of the
PPF as early as E13–14. They referred to the malformed tissue as the posthepatic mesenchymal plate or
the dorsal plate of the septum transversum. However,
when comparing the data, it appears that the tissue
being described in all of the studies is the PPF. The 3D
reconstructions provided in the present study allow for
a clear visualization of the regional defects within the
PPF. The malformed areas are consistently located in
the dorsolateral region. Correspondingly, the dorsolateral region of the diaphragm musculature is precisely
the area affected in CDH. Thus the data are consistent
with a causal relationship between a defect in the
initial PPF and later diaphragmatic musculature malformations. Furthermore, these data provide the focus
for future studies that may examine potential mechanisms underlying the normal and pathological formation of the dorsolateral PPF.
It should be stressed that the regions of the PPF that
are missing are not merely the somatic mesodermal
tissues that expand to close the pleuroperitoneal canal
(PPC). PPCs form dorsolaterally located channels that
link the future abdominal and thoracic cavities. The
failure of the PPC to close adequately is often cited as
the key pathogenic factor associated with CDH (16).
However, the defects are obvious before PPC closure,
and the regions of missing musculature extend well
beyond the PPC (2). It is apparent from the 3D reconstructions of defective PPFs that the degree to which
more medially and ventrally located tissues are affected varies. It is reasonable to hypothesize that the
variability in the size of the holes in the diaphragmatic
musculature with CDH is dictated by the extent of the
initial defect within the PPF.
There are a number of fundamental issues pertaining to the relationship between the PPF and CDH that
remained unresolved. First, do PPF defects result from
a problem with muscle precursor migration, proliferation, or differentiation? Alternatively, is there a problem with the formation of the underlying somatic mesodermal-derived connective tissue, on which the
muscle precursors develop? The fact that there is a
thickening of muscle fibers around the hole in the
diaphragm (2) is consistent with the hypothesis that at
least some of the muscle precursors normally destined
for the defective region of the primordial diaphragm
migrate around the missing substratum to occupy adjacent PPF tissue. Furthermore, the normal insertion
point for the phrenic nerve is often missing in defects
associated with the animal model of CDH. However,
the phrenic nerve is not absent, but, rather, its insertion point is translocated to a more ventrally located
region in the diaphragm musculature (2). This suggests that the PPF may be defective before the arrival
of the phrenic nerve (before E13.25) and that there is a
subsequent compensatory realignment of the nerve
contact point within the remaining PPF tissue. Studies
examining the earlier stages of normal and pathological PPF formation are underway to test these hypotheses.
A second issue of uncertainty is the potential role of
the liver in the pathogenesis of CDH. It is clear that the
liver occupies space normally reserved for the PPF
when there is a defect present. Whether the presence of
the liver tissue in this area is causal of the defect or
merely represents an early stage of liver displacement
cannot be clearly elucidated with the present data.
Third, there is predominance of left-sided defects in
infants with CDH that remains unexplained (16). The
ratio of left- vs. right-sided defects in the animal model
varies depending on which embryonic day the nitrofen
is administered to the dam (3, 5, 15). These data
suggest an age-dependent variation in the susceptibility of left and right PPFs to malformations. However, a
clear explanation for the laterality of the defect will
require further understanding of the etiology of CDH
or the mechanism of nitrofen teratogenicity.
PRIMORDIAL DIAPHRAGM DEFECTS IN CDH
This work was funded by the Medical Research Council of Canada,
March of Dimes, and Alberta Lung Association. J. J. Greer is a
Senior Scholar of the Alberta Heritage Foundation for Medical Research (AHFMR); D. W. Allan is a recipient of AHFMR and Killam
Studentship Awards; and R. P. Babiuk is a recipient of a Foundation
for Sudden Infant Deaths Studentship.
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