THE ANATOMICAL RECORD 294:633–644 (2011) Pleuroperitoneal Canal Closure and the Fetal Adrenal Gland SHOGO HAYASHI,1,2* YOSHITAKA FUKUZAWA,1 JOSÉ FRANCISCO RODRÍGUEZ-VÁZQUEZ,3 BAIK HWAN CHO,4 SAMUEL VERDUGO-LÓPEZ,3 GEN MURAKAMI,5 AND TAKASHI NAKANO2 1 Medical Education Center, Aichi Medical University School of Medicine, Nagakute, Japan 2 Department of Anatomy, Aichi Medical University School of Medicine, Nagakute, Japan 3 Departamento de Anatomı́a y Embriologı́a Humana II, Facultad de Medicina, Universidad Complutense, Madrid, España 4 Department of Surgery, Chonbuk National University School of Medicine, Jeonju, Korea 5 Division of Internal Medicine, Iwamizawa Kojin-kai Hospital, Iwamizawa, Japan ABSTRACT Pleuroperitoneal canal (PP canal) closure is generally considered to result from an increase in the height, and subsequent fusion, of the bilateral pleuroperitoneal folds (PP folds). However, the folds develop in the area ventral to the adrenal, in contrast to the ﬁnal position of the diaphragm, which extends to the dorsal side of the adrenal (the ‘‘retro-adrenal’’ diaphragm). We examined the semiserial histology of 20 human embryos and fetuses (crown-rump length 11–40 mm). We started observations of the canal at the stage through which the lung bud extends far caudally along the dorsal body wall to the level of the future adrenal, and the phrenic nerve has already reached the PP fold. Subsequently, the developing adrenal causes narrowing of the dorsocaudal parts of the canal, and provides the bilateral midsagittal recesses or ‘‘false’’ bottoms of the pleural cavity. However, at this stage, the PP fold mesenchymal cells are still restricted to the ventral side of the adrenal, especially along the liver and esophagus. Thereafter, in accordance with ascent of the lung, possibly due to anchoring of the liver to the adrenal, the PP fold mesenchymal cells seem to migrate laterally along the coelomic mesothelium covering some sheet-like loose mesenchymal tissue behind the adrenal. Final closure of the PP canal by lateral migration to provide the ‘‘retroadrenal’’ diaphragm is a process quite different from the common dogma. It is likely that the sheet-like loose mesenchymal tissue becomes the caudal part of the pleural cavity through a process involving cell death. Anat C 2011 Wiley-Liss, Inc. Rec, 294:633–644, 2011. V Key words: pleuroperitoneal canal; adrenal; lung bud; liver; coelomic mesothelium; human fetus Closure of the pleuroperitoneal canal (PP canal) is generally considered to result from an increase in the height and thickness, and even fusion, of the bilateral pleuroperitoneal folds (PP folds) containing the muscle precursors of the diaphragm (Hamilton and Mossman, 1978). According to some textbooks (e.g., Gray’s Anatomy; Collins, 2005), the bilateral pleural cavities extend into the mesenchyme on the ‘‘dorsal’’ side of the adrenal glands, the gonads and the mesonephric ridges. Thus, conversely, the PP canal is likely to be closed by the fusion of its edges, through growth of the organs C 2011 WILEY-LISS, INC. V Grant sponsor: Ministry of Health and Welfare (R & D program for Cancer Control), Republic of Korea; Grant number: 0620220-1. *Correspondence to: Shogo Hayashi, MD, PhD, Medical Education Center, Aichi University School of Medicine, Nagakute, Aichi 480-1195, Japan. Fax: þ81-561-63-1037. E-mail: shogo@ aichi-med-u.ac.jp Received 28 May 2010; Accepted 21 December 2010 DOI 10.1002/ar.21351 Published online 2 March 2011 in Wiley Online Library (wileyonlinelibrary.com). 634 HAYASHI ET AL. surrounding it, particularly that of the adrenal glands. However, as the fold develops in areas far cranioventral to the adrenal, i.e., those surrounded by the heart, liver, and cranial parts of the lung bud, does the PP fold really extend dorsocaudally to the dorsal side of the adrenal? Moreover, to our knowledge, the timing of closure of the PP canal has not yet been demonstrated in photographs. Botha (1959) has already provided a limited clue, as he reported that the PP canal is still widely open at the embryonic 12-mm stage and that muscle tissue extends into the membrane, which appears to close completely the canal at the 17.5-mm stage. Unfortunately, Botha did not demonstrate a sequence for the closure process, as this was beyond the scope of his study. Consequently, the aim of the present study was to re-examine the process of PP canal closure using semiserial sections of human embryos or fetuses. MATERIALS AND METHODS The study was performed in accordance with the provisions of the Declaration of Helsinki 1995 (as revised in Edinburgh 2000). We examined the parafﬁn-embedded histology of 20 embryos or fetuses [crown-rump length (CRL) 11–40 mm] at 6–12 weeks of gestation. However, as there was a possibility that estimates of ovulational age (coital age) might have been mixed with estimates based on the week of pregnancy, we classiﬁed these specimens into three groups according to the size: seven specimens with CRL 11–13 mm (early stage group), eight specimens with CRL 16–23 mm (mid-stage group), and ﬁve specimens with CRL 28–40 mm (late-stage group). All the specimens were part of the large collection kept at the Embryology Institute of the Universidad Complutense, Madrid, and were the products of miscarriages and ectopic pregnancies at the Department of Obstetrics of the University. Approval for the study was granted by the university ethics committee. Because of the nature of the specimens, we were unable to rule out the presence of pathology, but our aim was to describe the morphology that was commonly evident in each of the early, mid- and late-stage groups. After routine procedures for parafﬁn-embedded histology, most of the specimens were cut horizontally, and four sagittally (one specimen with CRL 19 mm; two with CRL 20 mm; one with CRL 21 mm) with a thickness of 5 lm, at intervals of 20 lm. Approximately 100–200 sections were prepared for each specimen, and all sections were stained with hematoxylin and eosin. RESULTS Early Stage Group (CRL 11–13 mm, Seven Specimens) The PP canal was identiﬁed as an irregularly shaped space surrounded by the liver, esophagus, mesonephros, and the so-called PP fold (Figs. 1–3). Likewise, through a narrow potential space between the lateral body wall and liver surface, the abdominal and thoracic parts of the coelomic cavity were connected (Figs. 1E, 2F, and 3F). This space was also included in the PP canal. The bilateral lung buds were surrounded by the liver from the ventral aspect, and not located on the cranial side of the liver, as is the case in adults (Fig. 1). Through the PP canal, the lung bud extended far caudally from the dorsal side of the heart, along the dorsal body wall, to the level of the future adrenal (Figs. 1–3). At this stage, the phrenic nerve had already reached the PP fold (Fig. 1A–C). In two of the seven early stage specimens (both, CRL 11 mm; Fig. 1), the mesonephros did not yet accompany the adrenal and was located closely to and facing the ventrally located PP fold. Most of the PP fold mesenchymal cells were restricted to the area between the heart and liver, especially along the dorsal aspect of the liver, but some of the cells formed a fold connecting to the dorsal body wall (Fig. 1C). In ﬁve specimens (Figs. 2 and 3), the mesonephros was attached to the lateral aspect of the adrenal. Moreover, the right adrenal was fused with the right lobe of the liver to provide a route for the future inferior vena cava (we have described the process of venous communication in another article: Jin et al., 2010). Thus, in these ﬁve specimens, the liver was anchored to the dorsal abdominal wall via the right adrenal. The PP fold mesenchymal cells were identiﬁed along the dorsal aspect of the liver, including a site close to the liver-adrenal fusion, and alongside the esophagus. The developing adrenal gland caused narrowing of the dorsocaudal part of the PP canal. During this process, bilateral ‘‘paramedian recesses’’ (blind sacs) of the PP canal or coelomic cavity were formed immediately caudal to the lung bud (Figs. 2E,F and 3D–F), being located between the bilateral adrenals and attached to them. These recesses appeared to form the ‘‘bottoms’’ of the canal, creating an impression that the primitive pleural cavity ended on or near the adrenal. However, the PP canal was still open bilaterally alongside the posterolateral surface of the liver, especially between the liver and the mesonephros, despite the fact that, at the craniolateral ends, the PP fold connected with the lateral coelomic mesothelium (Fig. 3A). Thus, the adrenal did not close the PP canal. Consistently on the right side (Fig. 1E) and often on both sides (three of the seven specimens; Figs. 2D and 3C,D), the esophagus accompanied a recess or recesses of the coelomic cavity (the pneumatoenteric recess described by Kanagasuntheram, 1957). The pneumatoenteric recess opened or communicated with the PP canal in the cranial part, whereas the right recess continued to the developing lesser sac at the caudal end. Thus, the right recess contained the developing caudate lobe of the liver (we have described the detailed morphology in another article: Hwang et al., 2010). The bilateral lung buds carried free caudal ends without a mesentery-like structure connecting to the esophagus (Figs. 1E, 2D, and 3C). On the dorsal side of the lung bud, thick, loose, sheet-like mesenchymal tissue was present along the dorsal body wall (Figs. 2 and 3). This sheet-like structure was covered by the coelomic epithelium. Notably, in four of the seven early stage specimens (CRL 11 or 13 mm), the PP fold connected with the covering mesothelium at the level of the heart apex (Figs. 2A,B and 3A). Mid-Stage Group (CRL 16–23 mm, Eight Specimens) In this group, we found several critical events involved in closure of the PP canal. However, the larger specimens did not always show the hypothetical later step of sequential events. One of the striking differences HUMAN PLEUROPERITONEAL CANAL CLOSURE 635 Fig. 1. Pleuroperitoneal canal opens in the caudal side: an 11-mm CRL-embryo. Horizontal sections. Hematoxylin and eosin (HE) staining. The right-hand side of the ﬁgure corresponds to the right side of the body. A: (E) is the most cranial (caudal) level in this ﬁgure. A: displays a lower magniﬁcation view, whereas B, C, D, and E are prepared at the same magniﬁcation (scale bars in A and B). The pleuroperitoneal canal is a narrow space surrounded by the bilateral lung buds (LB), the pleuroperitoneal fold (PPF), the mesonephros (MN), and the esophagus (E). In this specimen, the canal gives off two recess-like structures in A and C (open stars), but they open in the caudal side (B and D). The pericardial cavity (PCC) is closed in A. The phrenic nerve has already reached the caudal part of the PPF (arrows in A–C). E shows caudal ends of the lung bud without mesentery-like structure: the left end is caudal to the right one in this specimen. In E, the esophagus accompanies the pneumatoenteric recess of the coelomic cavity (black stars in D and E). The most lateral and caudal parts of the pleuroperitoneal canal are indicated by open circles in E. At any level, the liver (L) is still separated from the dorsal body wall by the coelomic cavity. AO, aorta; DV, ductus venosus; PCV, posterior cardinal vein. between this stage, and the early stage was in the topographical anatomy of the lung, adrenal, and diaphragm. The developing lung ascended to occupy a ﬁnal position alongside almost the entire craniocaudal extent of the heart. Thus, the lung disappeared from the craniocaudal level of the adrenal (Fig. 4; CRL 17 mm). However, as seen in Fig. 5 (CRL 23 mm), the caudal end of the lung bud still remained at the level of the adrenal. In a specimen with CRL 19 mm (Fig. 6), the lung was located far cranial to the adrenal. 636 HAYASHI ET AL. Fig. 2. Pleuroperitoneal fold mesenchyme contributes to the liver fusion with the right adrenal: a 13-mm CRL-embryo. Horizontal sections. HE staining. The right-hand side of the ﬁgure corresponds to the right side of the body. A: (F) is the most cranial (caudal) level in this ﬁgure. The pleuroperitoneal fold (PPF), containing dense mesenchymal tissues (asterisks), separates the liver (L) from the heart (H in A) and from the lung bud (LB in B). However, in C and D, the fold does not attach to the body wall, but the dense mesenchyme (asterisks) is restricted along the dorsal aspect of the liver near the esophagus (E). The dense mesenchyme contributes to fusion of the adrenal (AD) with the liver in E and F, but the inferior vena cava has not yet formed. The caudal part of the pleuroperitoneal canal makes the recess or blind sac attaching to the adrenal (open stars, E and F). The pneumatoenteric recess is seen in C–E (black stars). The left lung end is caudal to the right one in this specimen. Note a thick sheet of loose mesenchymal tissues (SLMT) in the dorsal and lateral sides of the lung bud. The most lateral and caudal part of the pleuroperitoneal canal is indicated by open circles in F. All panels are prepared at the same magniﬁcation (scale bar in A). AO, aorta; GC, gastric cardia; MN, mesonephros; PCV, posterior cardinal vein. The PP canal was closed by the diaphragm extending ‘‘behind’’ the adrenal. Within the developmental period between the early and mid stages, the PP fold mesenchymal cells appeared to reach the dorsal site immediately ventral to the abdominal aorta. As seen in Fig. 4, the PP fold mesenchymal cells were concentrated near the esophagus and appeared to be undergoing lateral migration along the coelomic mesothelium behind the adrenal. This mesothelium covered the ventral aspect of the sheet-like mesenchymal tissue along the dorsal body HUMAN PLEUROPERITONEAL CANAL CLOSURE 637 Fig. 3. False bottom of the pleural cavity near the adrenal: an 11mm CRL-embryo. A specimen different from that shown in Fig. 1. Horizontal sections. HE staining. The right-hand side of the ﬁgure corresponds to the right side of the body. A: (F) is the most cranial (caudal) level in this ﬁgure. The pleuroperitoneal fold (PPF), containing dense mesenchymal tissues, separates the liver (L) from the heart (H) in A, but it does not attach to the body wall in B. The fold attaches to the coelomic mesothelium in the cranial parts (arrows in A). The right lung end is caudal to the left one in this specimen (C and D). In D, E, and F, the adrenal (AD) is fused with the liver to provide a route of the inferior vena cava (IVC). The caudal parts of the pleuroperitoneal canal make the recess or blind sac attaching to the adrenal (open stars, D– F): they appeared to be the bottom of the pleural cavity, but the pleuroperitoneal canal is not closed. The most lateral and caudal parts of the pleuroperitoneal canal are indicated by open circles in panel F. The dense mesenchymal tissues are concentrated near the esophagus (E) (PPF in C). The pneumatoenteric recess is indicated by black stars in C–F. Note a thick sheet of loose mesenchymal tissues (SLMT) along the dorsal body wall. A, B, and F (C–E) are of the same magniﬁcation (scale bars in A and C). AO, aorta; GC, gastric cardia; LB, lung bud; MN, mesonephros; H, heart; PCV, posterior cardinal vein. wall (see the ﬁnal paragraph of the ﬁrst subsection). The celiac ganglion of the sympathetic nerve was located immediately caudal to the developing diaphragm. As shown in Fig. 5, the PP fold mesenchymal cells extended later- ally to reach the body wall, whereas the sheet-like mesenchymal tissue was reduced in thickness. The gastric cardia with the spleen, as well as the mesonephros and gonad, were located on the abdominal side 638 HAYASHI ET AL. Fig. 4. Pleuroperitoneal fold mesenchymal cells migrating along the mesothelium: a 17-mm CRL-embryo. Horizontal sections. HE staining. The right-hand side of the ﬁgure corresponds to the right side of the body. A: (C) is the most cranial (caudal) in the ﬁgure. B shows the most caudal part of the pleural cavity (PLC): the pleuroperitoneal canal was closed in this level. The pleuroperitoneal fold mesenchymal cells (PPF) are concentrated in a site behind the abdominal esophagus (GC in A) and between the bilateral adrenals (AD in B). The mesenchymal cells appear to migrate laterally along the mesothelium covering the thick sheet of loose mesenchymal tissue (SLMT). Black stars indicate the developing lesser sac containing the caudate lobe of the liver (CL). B and C are prepared at the same magniﬁcation (scale bars in A and B). AO, aorta; CG, celiac ganglion; GC, gastric cardia; IVC, inferior vena cava; L, liver; LGA, left gastric artery; MN, mesonephros. HUMAN PLEUROPERITONEAL CANAL CLOSURE Fig. 5. Diaphragm extending behind the adrenal: a 23-mm CRLembryo. Horizontal sections. HE staining. The right-hand side of the ﬁgure corresponds to the right side of the body. A: (C) is the most cranial (caudal) level in this ﬁgure. Arrowheads in A, B, and C indicate attachments of the developing diaphragm to the lateral body wall. D and F are higher magniﬁcation views of the central part of A and C, respectively. E (the caudal end of the lung) corresponds to an intermediate level between A and B. In A, the caudal parts of the lung bud (LB) are still located in the level of the liver (L) and right adrenal (AD). 639 The sheet-like loose mesenchymal tissues (SLMT in F) is thin or difﬁcult to identify in A–E. Instead, the pleural cavity (PLC) extends widely between the dorsal body wall and diaphragm (arrows). In D, the latter structure contains dense tissues, possibly muscle precursors, near the esophagus (E). A–C are prepared at the same magniﬁcation (scale bars in A, D, E, and F). AO, aorta; CL, caudate lobe of the liver; DV, ductus venosus; H, heart; PCV, posterior cardinal vein; E, esophagus; GC, gastric cardia; IVC, inferior vena cava; SP, spleen. 640 HAYASHI ET AL. the primitive pleural cavity was located at the level of the 12th thoracic or ﬁrst lumbar vertebra (Fig. 6). Most of this bottom area was covered by the adrenal from the ventral side. A possible remnant of the PP fold (Fig. 6B) was evident as a loose tissue band connecting the lung and diaphragm, being similar to the ligamentum pulmonale in adults. The paramedian recess on or near the adrenal (the false bottom of the pleural cavity) had disappeared in mid-stage specimens. Late-Stage Group (CRL 28–40 mm, Five Specimens) At this stage, the diaphragm was already situated in its ﬁnal position. The right adrenal and liver were separated by loose tissue (Fig. 8). The striated muscle ﬁbers of the diaphragm were clearly identiﬁed near and along the esophagus. However, muscle ﬁbers did not cross the ventral aspect of the esophagus. The dome-like cranial part of the diaphragm, covering the future bare area of the liver, was still membranous. The sheet-like mesenchymal tissue was reduced in thickness or difﬁcult to ﬁnd. Thus, the parietal pleura were located closely to the rib and intercostal nerve (Fig. 8). The left pneumatoenteric recess had disappeared, whereas the right recess (parts of the lesser sac) had expanded to contain the large caudate lobe of the liver. DISCUSSION Fig. 6. Muscle ﬁber distribution in the pleuroperitoneal fold: a 19mm CRL-embryo and a 21-mm CRL-embryo. Sagittal sections. HE staining. A specimen shown in A, B, C, and D is bit earlier than another specimen shown in E and F. A (D) is the most rightward (leftward) level in these four panels. The pleuroperitoneal fold extends along the cranial side of the liver (L) and bilateral adrenals. Muscle ﬁbers (arrows) are concentrated around the esophagus (E) as well as in the cranial side of the adrenal (AD) and the caudate lobe of the liver (CL). In B, a possible remnant of the pleuroperitoneal fold is seen (asterisks). All panels are prepared at the same magniﬁcation (scale bar in A). AO, aorta; CG, celiac ganglion; DV, ductus venosus; GD, gonad; H, heart; IVC, inferior vena cava; J, jejunum; K, deﬁnite kidney; LB, lung bud; P, pancreas; PV, portal vein; S, stomach; T, trachea. of the diaphragm extending behind the adrenal (Figs. 5 and 6). Notably, on both sides of the body, the caudal aspect of the diaphragm was connected tightly with the mesonephros (Fig. 7). The triangular-shaped bottom of The present study demonstrated paramedian recesses at the dorsocaudal part of the PP canal in fetuses with CRL 11–13 mm. These recesses contained the bilateral lung buds and ended on or near the developing adrenal. However, the lateral parts of the PP canal still maintained communication between the thoracic and abdominal parts of the coelomic cavity along the liver surface. Thus, these paramedian recesses provided false bottoms for the pleural or thoracic cavity. The bilateral adrenals did not close the PP canal by acting as obstacles. Likewise, we did not ﬁnd any direct contribution of the bilateral PP folds to closure of the PP canal, such as fusion of the crescentric free edges. Such morphology may be attributable to overinterpretation of fusion between the mesonephros and PP fold or between the PP fold and the lateral coelomic mesothelium. Instead, closure of the PP canal seems to be completed by lateral migration of the PP fold mesenchymal cells along the coelomic mesothelium behind the adrenal. Figure 9 shows the hypothetical courses of the PP fold mesenchymal cells. In short, they seem to migrate (1) caudally along the dorsal aspect of the liver, (2) dorsally along and around the esophagus, and (3) laterally along the coelomic mesothelium. In the early stage, the PP fold was located on the ventral side of the adrenal, gonad, and mesonephros. However, to separate these organs from the future pleural or thoracic cavity, the diaphragm should extend along the dorsal side of these abdominal viscera, as is the case for the adult pleural recesses. The most striking observation in this study was ‘‘switching’’ from the ventrally located PP fold to the ﬁnal, retro-adrenal diaphragm. This event seems to occur within a short period between the early and mid stages, that is, at the CRL 13–16-mm stage or possibly within a few days at an ovulation age of 6 HUMAN PLEUROPERITONEAL CANAL CLOSURE 641 Fig. 7. Diaphragm and mesonephros: a 19-mm CRL-embryo and a 20-mm CRL-embryo. Sagittal sections. HE staining. A and B: 19 mmCRL; C and D: 20-mm CRL. A and C (B and D) display the cranial end of the right (left) mesonephros (MN).The connective tissues of the meso- nephros continue to the diaphragm (DIA). The adrenal is located lateral side of these panels. All panels are prepared at the same magniﬁcation (scale bar in A). CG, celiac ganglion; GC, gastric cardia; GD, gonad; L, liver; S, stomach. weeks. The retro-adrenal diaphragm is likely to form along, or even replace, the coelomic mesothelium covering the loose, dorsal, sheet-like mesenchymal tissue. The connection between the covering mesothelium and the PP fold is ﬁrst evident alongside the heart even at the CRL 11-mm stage. However, the ﬁrst connection site was located ventral, lateral, and far cranial to the adrenal (Figs. 2B and 3A). To approach a site behind the ad- renal, a caudal course along and around the esophagus is most likely (Fig. 9). We hypothesize that the sheetlike mesenchymal tissue changes to the most caudal part of the pleural or thoracic cavity through a mechanism involving cell death. This change seems to be closely related to another marked change in the topographical relationship between the liver and the lung. Consequently, in humans, the bilateral PP folds 642 HAYASHI ET AL. Fig. 8. Final topographical anatomy of the diaphragm: a 35-mm CRL-fetus. Horizontal sections. HE staining. The right-hand side of the ﬁgure corresponds to the right side of the body. A (D) is the most cranial (caudal) level in this ﬁgure. In A and B, the diaphragm (DIA) is thick with striated muscles near the esophagus (E), whereas the lateral parts are membranous. A vacant pleural cavity (PLC) in B indicates a distance between the lung and adrenal. In C and D, the right adrenal (AD) does not attach to the liver (L) but is separated by a loose tissue (arrows). In D, the lesser sac (black stars) contains the caudate lobe of the liver (CL). The mesonephros disappears in these levels. A sheet-like mesenchymal tissue is difﬁcult to identify (cf. Fig. 4). All panels are prepared at the same magniﬁcation (scale bar in A). AO, aorta; GC, gastric cardia; ICN, intercostal nerve; IVC, inferior vena cava; LB, lung bud. themselves do not seem to fuse together to form a domelike diaphragm. We speculate that, in addition to the lateral plate mesoderm, the PP fold mesenchymal cells also contribute to fusion between the liver and the right adrenal in fetuses at the CRL 11–13 mm stage. Anchoring of the liver to the right adrenal surface seems to be critically important not only for development of the inferior vena cava (Jin et al., 2010) but also for ascent of the lung bud from the level of the adrenal. Without the liver alongside the heart, in turn, the lung seems to easily occupy in the future thoracic cavity. In the left side of the body, fusion between the adrenal and liver seems to be interrupted by the developing gastric cardia. Thus, conversely, the PP fold mesenchymal cells seem to simply migrate along the dorsal aspect of the liver, the esophagus, and the mesothelium covering the sheet-like mesenchymal tissue. In the late-stage group, the liver and right adrenal were clearly separated by loose tissue. Thus, the PP fold cells may play a limited, temporary role in fusion between the liver and the right adrenal. Using adult cadavers, Kawada et al. (2007) investigated the histological architecture of the small interface area between the pleural and peritoneal cavities without the diaphragm and other striated muscles: this area, or Bochdalek’s triangle, was consistently located just cranial to the 12th rib and often behind the kidney. Bochdalek’s triangle does not seem to correspond to the paramedian recess near the fetal adrenal but to the triangular-shaped bottom of the pleural cavity derived from the sheet-like mesenchymal tissue along the dorsal body wall. This bottom part seems to be uncovered by the deﬁnitive adrenal after birth. Bochdalek’s triangle in the adult may correspond to the site of initial attachment between the PP fold and the lateral coelomic mesothelium. Recently, Greer and coworkers have extensively described the pathogenesis of nitrofen-induced congenital diaphragmatic hernia (CDH) (Clugston et al., 2010a,b; Clugston and Greer, 2007): Clugston et al. (2008) identiﬁed human genes related to CDH and demonstrated that they are expressed in the nonmuscular, mesenchymal component of the diaphragm. Thus, CDH HUMAN PLEUROPERITONEAL CANAL CLOSURE Fig. 9. Schematic representation showing hypothetical migration courses of the pleuroperitoneal fold mesenchymal cells. In A, we superimpose thoracoabdominal structures of stages 11–23-mm CRL to show sequential migration processes of the pleuroperitoneal fold mesenchymal cells: (1) along the posterior surface of the liver (L) that faces the heart (H), (2) along the posterior surface of the liver that faces the lung bud (LB), (3) toward the dorsal aspect of the esophagus (E), (4) toward the fusion between the right adrenal (AD) and liver and, (5) along the mesothelium covering the bilateral sheet-like loose mes- 643 enchymal tissues (SLMT). The Process 5 or lateral migration does not ﬁnally occur but stars early possibly together with the Process 3: it is ﬁrst seen at the superior level above the adrenal (see Fig. 2B). The pleuroperitoneal fold mesenchymal cells are drawn by blue–green belt, but at a certain stage if limited, they never provide a continuous belt from the Processes 1–5. B and C are cross sections corresponding to those Processes 1–2 or 4–5, respectively. The gastric cardia (GC) is actually located behind the liver, but the former is pulled caudally to show the Process 5. AO, aorta. 644 HAYASHI ET AL. has its origins in a mesenchymal defect; Ackerman et al. (2005) reported that the lung and diaphragm might carry a common regulator gene such as Fog2, and that the lung bud is essentially important for enlargement of the canal; Belik et al. (2003) reported that epithelium-dependent airway muscle relaxation was signiﬁcantly decreased in CDH. These reports are not contrary to the present hypothesis that the PP fold mesenchymal cells greatly contribute to fusion between the right liver and the adrenal, rather than fusion between the bilateral fold edges themselves. Difﬁculty with lung ascent may lead to CDH as a result of defective lung development. It is well known that an abnormal communication persisting between the pleural and peritoneal cavities occurs more frequently on the left. 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