Pediatr Surg Int (2000) 16: 465472

Springer-Verlag 2000

MAIN TOPIC

S. W. Beasley J. Diez Pardo B. Q. Qi J. A. Tovar H. M. Xia

The contribution of the adriamycin-induced rat model of the VATER association to our understanding of congenital abnormalities and their embryogenesis
Accepted: 19 January 2000

Abstract The adriamycin-induced rat model of the VATER association has provided a means of studying the morphogenesis of a variety of major congenital structural abnormalities similar to those seen in humans with the VATER association. Most interest has been centered on the foregut, where the model has claried some aspects of the development of esophageal atresia (EA), tracheal agenesis, and other communicating bronchopulmonary foregut malformations. It has demonstrated aberrations in the nerve supply to the esophagus in EA and allowed the study of tracheomalacia. A relationship between an abnormal notochord, foregut abnormalities, and vertebral defects has been shown, and the model has reignited interest in the role of the notochord as a regional organizer of axial development. The normal temporospatial characteristics of apoptosis during fore- and hindgut development is disturbed in this model, resulting in abnormal morphology. The indications are that this model will continue to clarify the processes that lead to many of the structural congenital abnormalities that are seen in infants born with the VATER association. Key words Adriamycin Apoptosis Embryogenesis Esophageal atresia Notochord VATER association

Introduction
When the cytotoxic eects of adriamycin and its application in chemotherapy were rst being studied, relatively little research was undertaken to determine whether it had any teratogenic actions. One key study [1] mentioned en passant that some ospring of pregnant
S. W. Beasley (&) J. Diez Pardo B. Q. Qi J. A. Tovar H. M. Xia Department of Paediatric Surgery, Private Bag 4710, Christchurch Hospital, Christchurch, New Zealand

rats exposed to adriamycin had esophageal atresia (EA), but for many years pediatric surgeons and embryologists were largely unaware of this observation. The signicance that it provided an animal model for the study of foregut abnormalities was not realized until 1996, when Diez Pardo et al. published a preliminary report describing it as a new rodent experimental model of EA and tracheoesophageal stula (TEF) [2]. But the model had its problems: too much adriamycin administered too early in gestation led to death and resorption of virtually all embryos; too little too late failed to produce them. At rst, research was directed at determining the correct dose of adriamycin and the precise timing of its administration that would reliably produce EA [3]. Once the model had been rened, serious documentation of the abnormalities it produced commenced in earnest. It was soon recognized that the adriamycin-exposed rat fetus provided an animal model not only of EA, but also of other abnormalities similar to those seen in humans with the VATER association. Several research groups are now using this model to study EA/TEF [46], and it is anticipated that it may contribute to resolving some of the controversies and problems encountered during the management of the dicult clinical situations EA presents. Moreover, it is suspected that the presence of associated anomalies, such as those encountered in humans with the VATER (or VACTERL) or CHARGE associations, may have a common pathogenesis, but at this stage that remains largely unknown. Several observations point to the possible involvement of the neural crest in the origin of some components of these associations. Examples include the maturational dysautonomia and minor facial anomalies observed in some of these patients [7, 8] and the common embryological basis of many of the associated anomalies that are grouped under various acronyms [918]. This paper provides a brief summary of the role of the adriamycin-induced rat model (AIRM) of the VATER association in increasing our knowledge of these abnormalities and how they may occur.

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Materials and methods

All experiments using this animal model have been approved by local animal ethics or institutional research committees, and are performed in strict accordance with the European Union regulations for animal care (EEC 86/L609). In most published studies the types of animals used and the methodology employed have been strikingly similar [2, 46, 19 23]. Usually, accurately time-mated female Sprague-Dawley or Wistar rats weighing between 200 and 250 g receive intraperitoneal (i.p.) injection of 2 mg/kg adriamycin dissolved in sterile saline (0.5 mg/ml) on days 8 and 9 of gestation, or 1.75 mg/kg on days 69 inclusive. The nding of spermatozoa in an unstained vaginal smear after mating is designated as day 0 of gestation. Control rats receive saline only on the same days. A cesarean section is performed on day 21 of gestation, shortly before term. The fetuses are removed, killed, immersed in 10% formalin, and maintained for 7 days at 1820 C for adequate xation. Each animal is subsequently blotted, weighed, and examined under a binocular surgical microscope for external and internal anomalies. The primitive foregut Detailed studies of the events that occur during the critical period of foregut development (i.e., tracheoesophageal separation) have necessitated a reduction in the period of time-mating of the rats to within 8 h [24]. Pregnant rats are killed at 8-h intervals from day 11 to day 13 and their embryos recovered by cesarean section and xed in 10% buered formalin for 210 h, depending on their size. Embryos younger than day 13 of gestation are sandwiched in melted agar to facilitate orientation and handling [24, 25]. Embryos are sectioned transversely at 5 lm thickness, stained with hematoxylin and ewin (H&E), and examined serially. The foregut is studied from the 4th pharyngeal pouch to the lowest point of the respiratory primordium. The trachea Tracheomalacia (TM) can be induced by i.p. injection of adriamycin into timed-pregnant rats between days 6 and 9 of gestation [26]. Specic measurements of tracheal morphology on cross-section include length of cartilage, transverse length of the membranous trachea, anteroposterior (AP) and transverse diameters, tracheal cross-sectional area, and the number of tracheal segments. These measurements can be adjusted to correct for variation in crown-rump length. Derived measurements include the cartilage/ membranous ratio and the AP/transverse diameter ratio. Other studies of the trachea and bronchi have involved en-bloc removal of the larynx, trachea, lungs, esophagus, and stomach followed by a sequence of staining techniques [27, 28]. The heart and great vessels Study of the heart and great vessels involves transverse section of the fetuses [29]. Blocks obtained from the neck to the upper abdomen are processed, embedded in paran, and sectioned at a thickness of 5 lm. Sections are cut serially and every 10th section is studied, except in crucial regions where every 5th section is viewed. Alternatively, fetuses have been examined in situ for great-vessel anomalies and, after dissection, the heart is weighed and anomalies of the interatrial septum and atrioventricular valves are observed through the atria [30]. The heart weight can be expressed as a percentage of body weight. The heart is cut transversely in an equatorial plane midway between the apex and the aortic root to examine the interventricular septum, the right outow tract, and the subaortic portion. Macro-photographs of all malformations are taken.

The thymus and parathyroids The thymus is retrieved through dissection of the anterior mediastinum under a surgical microscope. Its weight is recorded as percentage body weight. Parathyroids, thyroid, and ectopic thymus are studied by embedding cervical blocks in paran: these are sectioned transversely every 3 lm in the horizontal plane and stained with H&E for histologic study [30]. The anorectum Similar techniques to those that have been used to study the foregut have been applied to the hindgut. The greatest technical diculties have related to obtaining correct orientation of the hindgut. The axial skeleton and cranium To study the axial skeleton, cranium, and limbs, fetuses are recovered by cesarean section on gestational day 21 and processed as described above. Instead of xation in formalin, they are eviscerated and the skin and soft tissues stripped. The carcasses undergo skeletal staining by modifying the techniques of Simons [31] and Ojeda [32]. First, they are xed in alcohol-Alcian blue solution, dehydrated in absolute alcohol, and macerated in 1% KOH-alizarin red solution. The specimens are then immersed in a cleaning solution of glycerol and KOH that is progressively changed into pure glycerol by the addition of successive daily increments. The material is then stored for observation under transillumination with a surgical microscope at 1640 times magnication. The scarce remaining soft tissues become translucent, whereas the cartilage is stained blue and the bone is purple-red. The entire skeleton can be inspected, the number of vertebral segments counted, and any anomalies noted and photographed. The number of skeletal pieces and the incidence of bony malformations in control and adriamycin-treated fetuses are compared. Synovial joints are examined by serial histologic section [33]. Three-dimensional (3D) reconstructive techniques Williams et al. [34, 35] have rened a technique for creating ``skinned'' 3D images of minute structures during early embryogenesis in the rat. This technique has vastly improved the ability to visualize the morphologic changes that occur during organogenesis. The embryos are harvested and processed at specic gestational ages as described earlier. Digitized photographs taken of serial sections imported onto a computer with the boundaries of the key structures traced and the background removed allow sequential 3D layering according to the degree of separation of the initial sections [34]. The images can be aligned in relation to the neural-tube oor plate and ``skinned'' to produce a solid 3D reconstruction that can be viewed from any angle. This technique has been used to demonstrate the course of the notochord in relation to the mesenchyme and epithelium of the foregut structures, enabling accurate and unbiased interpretation of a large number of serial sections and conveying information that would otherwise be dicult to visualize.

Results
The normal rat Determination of abnormal development demands a thorough knowledge of normal embryogenesis in the animal model employed, in this case the rat. For this reason, the AIRM of the VATER association has led to several publications describing normal organogenesis

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[24, 25], for without this information valid comparisons cannot be made. An example relates to the abnormal cartilaginous rings that occur in TM, a frequent accompaniment of EA/TEF in both humans and the adriamycin-exposed rat. It might be tempting to consider any aberration of the classical ``c''-shaped tracheal ring to represent an abnormality, but careful analysis of the normal rat has shown that minor imperfections of tracheal rings (e.g., bid rings) are extremely common, and as such cannot be considered abnormal [27]. Only once the imperfections of tracheal rings in the normal rat were fully documented was it possible to fully appreciate the more severe and bizarre abnormalities seen in those that develop EA or tracheal agenesis (TA). An unexpected sequel to the rat model of EA and the VATER association has been the impetus it has provided to the study of normal embryogenesis, particularly of the foregut. When the initial studies of foregut development were performed [36, 37], the role of apoptosis in organogenesis was not suspected. Most research into apoptosis has related to cell behaviour in tumors [38, 39], but it is now known that programmed cell death, as distinct from cell necrosis, plays an important role during embryologic morphogenesis as well. Studies have been performed in which the role of apoptosis in early foregut development has been claried [25], including recognition that it is the mechanism by which the trachea and esophagus separate following development of the bronchial buds. The spatial and temporal characteristics of the process have exposed the limitations of previous theories of tracheoesophageal separation [24], such as the ``tap water theory'' proposed by O'Rahilly and Muller [37]. More recently, it has become evident that a similar process may occur during urorectal separation [40]. Esophageal atresia This animal model of EA produces an appearance almost identical to that seen in the human. The vast majority of rats have a blind upper esophageal pouch and a distal TEF communicating with the trachea near the carina [2]. In severe cases, it also produces TA and other communicating bronchopulmonary foregut malformations (CBPFM) [23]. At day 21 of gestation the EA is fully developed, as might be expected. However, if the rats are killed between gestational days 11 and 13 the abnormality can be observed as it develops. Clinicians involved in the management of infants with repaired EA are aware of the esophageal dysfunction that persists throughout childhood and into adult life [41, 42]. Whether esophageal dysmobility and poor esophageal clearance are related primarily to damage to the vagi during surgery or to an inherent problem with the innervation of the esophagus in EA has been uncertain. This model has helped demonstrate an inherent abnormality of the neurologic supply of the esophagus. One study of the course and relations of the vagus in 21

fetuses with EA showed inherent abnormalities in the course and branching pattern of the vagus nerves and deciencies in the extrinsic nerve-ber plexus of the lower esophagus [26]. Subsequent studies have demonstrated abnormalities of the neurotransmitters within the esophagus [20, 43]. Tracheal agenesis and CBPFM TA represents the extreme end of severity of tracheoesophageal abnormalities. The respiratory primordium appears to be more resistant to the insult of adriamycin than the esophageal primordium, in that 82% of fetuses developed EA whereas only 15% had TA [22]. Where the proximal foregut develops into trachea there is no esophagus in that region, and vice versa. Moreover, the severity and increased frequency of associated abnormalities in TA would indicate that it is the more severe abnormality [22]. The primitive foregut endoderm maintains its respiratory potential and can be induced by bronchial mesoderm to form a lung bud and to branch thereafter [44, 45]. This would account for the range of CBPFM seen in the adriamycin-exposed rat fetuses, and provide further evidence for the assertion that CBPFM and EA are variations of a spectrum of abnormalities with a common etiology [23]. The exact pathogenesis of TA remains uncertain, but may relate to ventral shift of the plane of tracheoesophageal separation [46, 47]. Further study of the timing and location of apoptosis of embryos developing TA may clarify this issue. Tracheomalacia The morphologic similarities between this model of TM and the description of human infants with TM is remarkable [48, 49]. The length of the cartilage is markedly reduced and the length of the membranous trachea increased, as in humans. The total length of the trachea in rats with EA is longer than in control rats [27, 28]. The cartilage/membranous ratio is reduced from 4.3 in the normal rat to 1.5 in EA [26]. The tracheal cartilages themselves are often grossly distorted and damaged, particularly in the proximal trachea [27, 28]. The combination of a broadened membranous trachea with a shortened or grossly abnormal ring of cartilage and consequent loss of the normal rigid support of the tracheal wall accounts for why the trachea becomes accid and collapses with positive intrathoracic pressure, leading to upper-airway obstruction [2628, 48]. Cardiovascular anomalies Qi et al. [20] compared the normal anatomy of the heart and great vessels in control fetuses at term with that of 36 adriamycin-treated fetuses. The latter had numerous cardiovascular malformations (CVM) including patent

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ductus arteriosus (PDA), ventricular septal defects (VSD), atrial septal defects (ASD), right aortic arch (RAA), and retroesophageal subclavian artery (RESA). RAA was common. Double aortic arch (DAA), separate carotid artery (SCA), and aortic coarctation (COA) were found only in EA specimens. Control animals consistently have normal cardiovascular systems [29, 30], whereas 65% of fetuses with EA have either heart or great-vessel malformations, or both. The association of EA and CVM is signicant (Fisher, P < 0.01) because 45 of 69 fetuses with EA had such anomalies compared with only 10 of 40 (25%) adriamycin-treated fetuses without EA (Table 1). The weight of the heart in EA fetuses was greater than in controls and in animals without EA. Anomalies of the great vessels are more frequently seen, such as RAA with left ductus arteriosus (21.7%) and aberrant right subclavian artery (ARSA) (13%). These vessels may create a complete vascular ring around the trachea, as do the less frequent DAA and RAA with right ductus arteriosus (RDA) with or without an aberrant subclavian artery. Thymus and parathyroids The thymus is absent in about 52% of adriamycin-treated rats with EA, and is grossly hypoplastic or ectopic in the remainder. Ectopic organs may be found in very aberrant locations. In 9 of 12 fetuses exposed to the teratogen but without EA a very small thymus was seen [30]. The normal rat has two parathyroid glands that are consistently located in the lateral surface of the thyroid glands. Almost all adriamycin-exposed fetuses without EA had
Table 1 Cardiac and great-vessel defects in rat fetuses exposed to adriamycin no-EA (n = 40) Heart defects Ventricular septal defect Narrow-outow pulmonary tract Tetralogy of Fallot Double-outlet right ventricle Truncus arteriosus Atrial septal defect Valve dysplasia Aneurysm of the sinus valsalvae Great-vessel defects Right aortic arch Aberrant right subclavian artery Double aortic arch Right ductus arteriosus Patent embryonal vessels Aortic coarctation 1 (2.5%) 1 (2.5%) 0 0 0 2 (5%) 1 (2.5%) 0 6 (15%) 0 0 0 0 0 EA (n = 69) 8 (11.6%) 6 (8.7%) 5 (7.2%) 3 (4.3%) 2 1 1 1 (2.9%) (1.4%) (1.4%) (1.4%) Total (n = 109) 9 (8.2%) 7 (6.4%) 5 (4.6%) 3 (2.7%) 2 3 2 1 (1.8%) (2.7%) (1.8%) (0.9%)

normal glands on both sides. The parathyroids were absent in 16/23 (69%)-animals with EA, and 4 animals had only one gland. An ectopic gland dorsal and cranial to the thyroid was located in 1 rat. All 23 fetuses with EA had an abnormal thymus (12 absent, 9 grossly hypoplastic, 2 ectopic) and 73% of these had associated CVM. Vertebral malformations Kotsios et al. [50] found multiple bone anomalies affecting the vertebrae, ribs, limb bones, and tail in 54% of EA fetuses exposed to adriamycin. Xia et al. [51] observed spinal malformations at some level in all rats with EA and in 39% of those without this malformation in a series of 88 adriamycin-exposed fetuses. No gross cervical-spinal malformations were seen, but there is normally a paucity of ossication at this level in the day 21 fetus. The thoracic spine was most frequently aected, particularly at the T2T12 levels, but more than 1 level was generally involved. The most common anomaly was a buttery vertebra with two independent ossication nuclei instead of one. Hemivertebrae and lack of fusion of the spinous apophyses was also encountered. Some fetuses appeared markedly lordotic, centered in the upper thoracic spine in 7, lower thoracic in 3, or lumbar in 1: this occurred even in the absence of demonstrable wedged vertebrae. The pelvis was small, and caudally-convergent ishiopubic rami and a lack of sacral vertebrae were seen in 27 adriamycin-treated fetuses. The number of caudal elements was dicult to ascertain because of their incomplete ossication at this stage, but was undoubtedly fewer in EA fetuses compared with control fetuses. Fetuses with anal atresia consistently had fewer than 5 sacral elements. Normal rats have 13 pairs of ribs attached to the thoracic vertebrae: 7 of these are true ribs fused to the sternum at their anterior end and 6 are unattached, oating or false ribs. This normal costal arrangement was observed in adriamycin-treated fetuses without EA, but 2 of 56 with EA had 6 true and 7 oating ribs. One fetus had 2 bayonet-shaped, malformed ribs and a malformed sternal manubrium was observed in 3 instances [51]. Hindgut development Interpretation of the morphologic changes that occur during hindgut development has proved more dicult than that of the foregut. However, apoptosis does appear to play a role during regression of the tailgut and urorectal separation [40]. Notochord Merei et al. [52] and Possoegel et al. [4] found that the notochord failed to separate from the foregut at the correct time, i.e., before gestational day 11. At day 11.5 the notochord was still attached to the foregut at one or

15 (21.7%) 9 (13%) 3 (4.3%) 3 (4.3%) 2 (2.9%) 1 (1.4%)

20 (18%) 9 (8.2%) 3 (2.7%) 3 (2.7%) 2 (1.8%) 1 (0.9%)

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more points instead of descending immediately ventral to the neural tube. Later, the notochord separated from the foregut to lie outside the developing sclerotomes. These authors found that by day 14 the notochord still had an irregular shape within the sclerotomes, and was associated with abnormal separation of the sclerotomes. More detailed investigation of the notochord in the embryo developing EA or TA has revealed major aberrations in the course and appearance of the notochord that correlate reasonably closely with the associated regional abnormalities that occur [21, 34]. In day 11 adriamycin-treated embryos the notochord remains attached to a foregut that is markedly narrowed, and on some occasions even occluded. On day 12 its adherence to the foregut extends for a variable distance between the primitive pharynx and a level proximal to the bronchial buds, at which point it becomes thickened. The foregut usually loses its continuity where it is in contact with the notochord. The embryo develops EA or TA when the notochord is grossly abnormal [21, 34]. Abnormal thickening of the notochord caudal to the termination of its ventral mesenchymal branch is evident in embryos with complete disruption of the foregut structures [34]. Limb abnormalities Adriamycin causes delayed bony growth and calcication. Malformations are seen in 61% of animals with EA and involve the humerus, radius, ulna, and to a lesser extent the tibia and bula [50, 33]. The bones are short, thick, and crooked, although all segments are complete except for the absence of a forelimb digit observed in 1 rat. One animal had sirenomelia. The cranium was normal in all specimens. These ndings are similar to the vertebral and limb malformations described in human babies with the VATER association [50]. Synovial joints appear unaected.

common, again as in humans. Only once has an isolated TEF without EA been reported [59], although this may be due in part to the dosage of adriamycin used. Secondly, the animal model reproduces most of the associated malformations found in EA patients, including gastrointestinal (GI) [2, 19], urologic [19], skeletal [50, 51], and cardiovascular [29]. Even more subtle aberrations of normal anatomy, such as vagal anatomy [5] and aberrations in the intrinsic innervation of the esophagus [20, 43], have been recorded: these are similar to those previously described in EA patients [60, 61]. Vertebral malformations resemble those found in humans, e.g., hemivertebrae. The VATER association In a very short time, this animal model has established itself as being able to reproduce reliably all the associated malformations seen with EA in the now well-recognized pattern of the VATER association. Children born with EA have a reported incidence of musculoskeletal defects ranging between 2% and 43% [6264]. Stevenson [65] observed extra vertebrae in 75% of patients with EA. Skeletal malformations are relatively more frequent when EA and anorectal atresia occur in combination [66]. Scoliosis is more likely when there are vertebral malformations (47%), and tends to be more severe in these patients than in those without hemivertebrae (14%) [67]. In duodenal atresia, a range of frequencies (2.1%37%) of vertebral malformations has been reported [68]. An association of vertebral defects with GI atresia is not surprising since both systems develop at a similar time, and are dependent on the notochord. Esophageal, duodenal, and anal atresia develop in sites that are in close proximity to the vertebral column and the notochord. Irrespective of the methodology employed [29, 30], a wide range of CVM have been identied. Abnormalities predominantly derive from abnormal conotruncal septation and rearrangement of the pharyngeal arteries (perimembranous VSD, narrow-outlet pulmonary tract, tetralogy of Fallot, double-outlet right ventricle, TA, RAA, ARSA, DAA, and RDA), consistent with a mechanism related to neural-crest development. Moreover, neural-crest-derived CVM are associated with either absence or anomalies of the pharyngeal arch-derived thymus and parathyroids, perhaps indicating a role for the neural crest in the pathogenesis of these malformations. Conotruncal septation and pharyngeal arch development take place in the rat between gestational days 10.5 and 12.5, and neural-crest-cell migration starts shortly before that time, while tracheoesophageal separation occurs between gestational days 11 and 12. For these reasons, the timing of prenatal exposure to the teratogen in our experiments would seem to be crucial in inducing foregut, cardiovascular, thymic, and parathyroid anomalies, although the exact mechanisms involved remain unclear.

Discussion
The prognosis of babies born with EA and TEF has progressively improved in recent decades [53] such that associated malformations (particularly those of the cardiovascular system and chromosomes) are now the most important determinants of survival [5457]. Future improvements in our understanding of EA and its management will derive from both laboratory investigations using the adriamycin-induced fetal rat model (AIRM) and careful analysis and review of the patients we treat, particularly those with problematic clinical situations [58]. There has been intense interest in the AIRM of EA for several reasons. First, it accurately and consistently reproduces the anatomy seen in the human infant with EA: the type of atresia most commonly identied is the same as the most common type in humans (Gross type C) [2]. Other variations are much less

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The notochord The observation of an abnormal notochord in embryos developing EA has excited considerable interest, in part because the notochord is believed to act as a primary organizer of axial structures (neural tube, sclerotomes, myotomes) during early embryogenesis [69, 70]. Abnormal development of the notochord may lead to a variety of congenital abnormalities [7173] including hemivertebrae, neurenteric remnants, GI duplications, and splitnotochord syndrome. Two recent studies [21, 34] have demonstrated signicant abnormalities of the notochord occurring in association with EA and TA. These embryos have persistent and abnormal adherence of the notochord to the foregut, and the notochord branches abnormally as it enters the foregut mesenchyme and draws the upper foregut in a dorsal direction. The severity of the foregut abnormality correlates well with the length of ventral extension of the notochord within the foregut mesenchyme. TA is associated with the greatest degree of notochord abnormality [22, 34]. The notochord becomes thickened when it has no foregut structures anterior to it, and loss of foregut continuity appears to occur when the notochord descends in the foregut mesenchyme. This leads to the speculation (as yet unproven) that the notochord may be involved in negative biochemical feedback mechanism with the foregut [34]. Tracheomalacia This animal model may have helped clarify whether TM is a primary or secondary abnormality. Benjamin et al. [74] considered the accidity of the trachea to be congenital, whereas Davies and Cywes [75] have postulated that TM is due to extrinsic compression by a dilated and hypertrophied upper-esophageal pouch that has adversely aected maturation of the tracheal wall. The presence of TM in the rat model in the absence of an adjacent upper pouch makes the latter theory unlikely. However, the model has also shown that extrinsic compression can exacerbate the severity of TM locally. For example, in the region of a DAA the disruption of the tracheal cartilage is worse than above or below that level. It may be that in the human a dilated upper esophageal pouch may worsen the eects of pre-existing TM, but is not the original cause [26, 49]. Tracheoesophageal separation One of the most important contributions of this animal model of the VATER association has been to renew interest in the processes of normal organogenesis. Correct interpretation of the morphologic changes that occur in the embryo developing EA has required reexamination of the processes that occur in the normal rat. The key question has related to how the trachea

separates from the primitive foregut, for which there has been no consensus up to now. Zaw-Tun [76] questioned the signicance of the tracheoesophageal septum (TES), or whether it even exists. He could nd no evidence of an ascending TES, the mechanism that was presumed to be responsible for separation of the trachea from the esophagus. There has also been controversy about the two lateral epithelial ridges, which were presumed to grow actively toward each other and fuse to partition the foregut into trachea and esophagus [37, 77, 78]. It is clear that in the rat embryo both bronchial buds appear before tracheal separation commences, i.e., when the trachea still shares a common lumen with the esophageal part of the primitive foregut [25]. This observation is not consistent with the popular ``tap water'' theory proposed by O'Rahilly and Muller [37], which describes how the lung bud grows caudally into the ventral foregut mesenchyme before dividing into primary bronchi. The process of bronchial budding that occurs prior to the TES has been claried by applying 3D reconstructive techniques to these embryos [35], a technique that also allows quantication of relative growth rates [35]. It would seem that tracheal separation involves three consecutive stages: (1) formation of bronchial buds from epithelial proliferation; (2) initiation of separation of the trachea and esophagus by epithelial apoptosis; and (3) completion of the separation process by epithelial proliferation [24]. Apoptosis It has been long known that dierential rates of cell growth and progressive cell dierentiation are key processes that determine the nal morphology of many organ systems. More recently, apoptosis has been identied as a third process that plays a role in the embryogenesis of some structures, including the central nervous system and digits [7982]. Apoptosis, or programmed cell death, is now recognized as being as much a part of embryonic development as are cell proliferation and dierentiation [83]. A recent study of normal rat embryogenesis has shown that a specic and consistent pattern of apoptosis appears to play a part in the early separation of the foregut into trachea and esophagus [25]. In day 12 embryos there is obvious debris of dead epithelial cells and their condensed nuclei (apoptotic bodies) at the junction of the attened epithelium posteriorly and the more prominent (respiratory) epithelium anteriorly. This corresponds to the location of the previously described TES. Apoptosis is always seen at a specic time and a specic location, i.e., it follows a characteristic temporospatial pattern, as part of a highly regulated process. Focal apoptosis contributes to morphogenesis and is under the precise control of genetic and local factors [84]. It appears that there may be a specic pattern of programmed cell death around the junction of the future tracheal and esophageal epithelium, an occurrence that

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contributes to normal separation of the trachea and esophagus. Apoptosis has also been observed during early development of the cloaca, and may be involved in the process of regression of the tailgut and urorectal septation [40]. In conclusion, the adriamycin-induced rat model of the VATER association has enabled study of the pathogenesis of a variety of congenital abnormalities that show a remarkable similarity to those seen in human infants who have the VATER association. It has also renewed interest in the processes of normal organogenesis, including the role of apoptosis in the determination of ultimate morphology, and the role of the notochord as a regional organizer.

17. 18. 19. 20.

21. 22.

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