Seminars in Pediatric Surgery (2010) 19, 201-208

Embryology of anorectal malformations

Dietrich Kluth, MD, PhD
From the Department of Pediatric Surgery, University Hospital, Leipzig University, Leipzig, Germany. KEYWORDS
Normal anorectal development; Embryology; Abnormal development; Animal models; Cloaca; Hindgut Today, the normal and abnormal development of the hindgut is still a matter of speculation. However, as the result of recent studies in appropriate animal models, most embryologic events that nally lead to abnormal hindgut development are better known than in the past: (1) the process of maldevelopment starts in early embryonic stages; (2) the cloacal membrane is always too short in its dorsal part, thus, the dorsal cloaca is missing; and (3) as a result, the hindgut remains attached to the sinus urogenitalis, forming the recto-urethral stula. In the past, an impaired process of septation was believed to be the main cause of abnormal hindgut development. In contrast to this, our results indicate that the development of the septum is more passive than active. Furthermore, the results of our studies in normal and abnormal development indicate that (1) the embryonic cloaca never passes through a stage that is similar to any form of anorectal malformation in neonates, including the so-called cloacas in female embryos, and (2) to explain abnormal development, studies in abnormal embryos are mandatory. 2010 Elsevier Inc. All rights reserved.

Despite many efforts, the embryology of numerous congenital anomalies in humans is still a matter of speculation because of the following: 1. A shortage of study material (both normal and abnormal embryos). 2. Various technical problems (difculties in the interpretation of serial sections, shortage of explanatory threedimensional reconstructions). 3. Misconceptions and/or outdated theories concerning normal and abnormal embryology. Fortunately, many animal models are known today that allow advanced embryologic studies in various embryologic elds. Especially for the studies of anorectal malformations (ARMs), several animal models exist. However, appropriate and illustrative descriptions in various elds of embryology are still lacking, which explains why today many typical
Supported in part by Deutsche Forschungsgemeinschaft, Grant Number KL 596/1, Hamburg Werner-Otto-Stiftung. Address reprint requests and correspondence: Dietric Kluth, MD, PhD, Department of Pediatric Surgery, University Hospital, Leipzig University, Liebigstra 18, 04103 Leipzig, Germany. E-mail address: dietrich.kluth@medizin.uni-leipzig.de.

malformations are still not sufciently explained. Pediatric surgeons are still confused when they are confronted with the embryologic background of normal and abnormal development. For misconceptions and/or outdated theories, Haeckels biogenetic law1 is one example. According to this theory, the human embryo recapitulates in its individual development (ontogeny) the morphology observed in all life forms (phylogeny). This means that during its development, an advanced species is seen to pass through stages represented by adult organisms of more primitive species.2 This theory has still an impact on the nomenclature of embryonic organs. This explains why human embryos have cloacas like adult birds and branchial clefts like adult sh. Another very popular misconception is the theory that malformations actually represent frozen stages of normal embryology (Hemmungsmibildung).3 As a result, our understanding of normal embryology stems rather from the interpretations of observed malformations than from proper embryologic observations. The theory of the rotation of the gut as a step in normal development is a perfect example for this misconception.

1055-8586/$ -see front matter 2010 Elsevier Inc. All rights reserved. doi:10.1053/j.sempedsurg.2010.03.005

202 The purpose of this work is to illustrate what we have learned from our studies in normal and abnormal hindgut development performed in our laboratory. We used scanning electron microscopy (SEM) to illustrate our ndings.4 In our experience, SEM allows the documentation of 3-dimensional embryonic structures in superior detail.5-7

Seminars in Pediatric Surgery, Vol 19, No 3, August 2010 resulted in a spectrum of ARMs as seen in humans. In principle, all these models are sufcient to study the embryology of abnormal hindgut development. Interestingly, all animal models presented with a similar spectrum of abnormalities.

The etiology of anorectal malformations

The etiology of ARMs is still unclear. Most researchers assume that its etiology is multifactorial. However, recently a number of animal models has been deployed to study the background of ARM. In these models genetic as well as environmental factors were identied. 1. The SD-mouse model: these mice, rst bred by Danforth,8 prominently feature a short tail and therefore also are known as Danforths short tail mice.9 2. The pig model: another famous model for abnormal hindgut development is the pig.10-12 3. The adriamycin model: in 1978, Thompson et al13 used adriamycin to induce various anomalies in high percentages in rat fetuses. Observed were esophageal atresias with tracheoesophageal stulas and ARMs amongst others. This model of the VACTERL association has been studied in detail by numerous research groups.14-18 Other substances that have proven to be useful in inducing ARM (mainly in rats) are etretinate,19,20 all-trans retinoic acid,21-23 and ethylenethiourea.24-27 Recently, Kim et al28 and Mo et al29 studied malformations in Gli mutant mice and found a spectrum of abnormalities similar to those in the VACTERL association. They could demonstrate that a modulation of the defective gene

Normal embryology of the hindgut

The normal embryology of the hindgut always has been a matter of debate because observations made in normal embryos should not only explain the normal embryology but also its abnormal counterpart. As a result, the explanation of normal embryology was always done with abnormal development in mind. Two major theories exist to explain the differentiation of the hindgut into the urogenital (ventral) and anorectal (dorsal) part: 1. The theory of the septation of the cloaca; and 2. The theory of the migration of the rectum. The latter had been modied by van der Putte10 in 1986. Another controversy exists of whether the urorectal septum fuses with the cloacal membrane (CM) in normal development or not.

Early development of the hindgut

The anorectal septum of the hindgut In very young embryos, the hindgut is a simple structure (Figure 1). Cranially, it is in continuity with the midgut; caudally, it is in direct contact with the ectoderm, thus forming the cloacal membrane. When development progresses, the caudal part of the hindgut, the cloaca,

Figure 1 Schematic drawing of normal cloacal development in rats (drawn after SEM photographs). (A) A 12.5-day embryo; (B) 14-day embryo; and (C) 15-day embryo. Note the movement of the cloacal membrane (CM) from a vertical to a horizontal position. This movement is caused by the ventral outgrowth of the genital tubercle and the cloaca. Note the descent of the urorectal fold (short arrows). The dorsal part of the cloacal membrane (gray dots) is the area of the future anal opening. Arrows with asterisk (*) point to the tail groove. This area is the xed point in development of the cloaca. HG, hindgut; CM, cloacal membrane; C, cloaca; TG, tail gut; A, allantois; S, sinus urogenitalis; W, Wolfan (mesonephric) duct; U, ureter.

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Embryology of Anorectal Malformations

203 The SEM was used because of the following advantages: 1. Serial sectioning of embryos and time-consuming 3-dimensional reconstructions are not necessary. 2. The embryo can be studied in all 3 dimensions on-line. 3. The images and photographs are of superior quality. The essential ndings of our study are summarized in Figure 1. In contrast to earlier reports, we found that (1) a septation of the cloaca by fusion of the lateral folds does not take place and (2) a migration of the anal opening or a shifting of the dorsal cloaca cannot be observed. The early cloacal development The starting point of this series is the cloaca in a 12.5-day-old rat embryo (Figure 2). At this stage, all features of a typical cloaca are present: the hindgut (HG) enters the cloaca (c) from dorsocranial whereas the allantois (a) (the forerunner of the bladder) can be identied as a cranioventral diverticulum. Between this diverticulum and the hindgut, the urorectal fold (arrow) can be seen. This fold marks the cranial border of the undifferentiated hindgut, the so-called cloaca. The mesonephric duct (Wolfan duct; W) enters the cloaca in its cranial part but in a relatively dorsal position. Caudally, the cloaca continues directly into the tailgut (TG). The CM extends in a slight concave curve from the caudal

differentiates into 2 separate organ systemsthe urogenital tract and the anorectal tract. Since the work of Tourneux30 and Retterer31 at the end of the 19th century, it has been generally accepted that the normal development of these tracts depends on the proper subdivision of the cloaca by a septum, the so-called urorectal septum. According to this theory, abnormal septal development always should result in abnormal cloacal development. However, there is no agreement among investigators about the nature of this septum and the way it develops. Although Tourneux30 thought that the septum moves down from cranial to caudal like a French curtain, Retterer31 speculated that lateral folds or ridges appear in the lumen of the cloaca. These ridges should fuse and thus form the septum, beginning cranial and ending caudal at the level of the CM. In the past, numerous investigators supported one of these theories. Stephens32 combined both theories, believing that this could best explain the various forms of ARMs. He claimed that the cranial part of the septum should grow downward as explained by Tourneux, whereas in the caudal part lateral ridges should fuse to form the septum in this area. In 1986, van der Putte10 denied the major role of the urorectal septum in the process of cloacal differentiation. The migration of the rectum Studying the morphology of ARM in human newborns, Bill and Johnson33 and later Gans and Friedman34 stated that in most forms of ARM the stula may present an ectopic anal opening. They concluded from these observations that the rectum actually migrates during normal development, from a rather high position to the normal area of the anal opening. If this process of migration stops before the anus has reached its denitive position in the area of the perineum, an ectopic anal canal would result. Although this speculation is rather attractive, neither these investigators nor other researchers were able to show any embryologic evidence of this migration. The shift of the dorsal cloaca In 1986, van der Putte10 modied the theory of a rectal or anal migration. While studying normal and abnormal pig embryos, he speculated that a shift of the dorsal cloaca takes place. This shift should bring the dorsal cloaca down to the area of the tail groove, thus establishing here the future anal opening.

Studies in the normal embryology of the hindgut (authors studies)

In 1995, we studied hindgut development in a series of staged rat embryos between the 10th and the 15th gestational day (comparable with human embryos between the third and 7th week of gestation and rat embryos between the 11th and 16th gestational day)4 by using SEM. A total of 245 embryos were analyzed in this study.
Figure 2 SEM photograph of the cloaca of a 12.5-day-old rat embryo. Lateral view of the cloaca after microdissection. The mesenchyme has been removed. See text for details. C, cloaca; HG; hindgut; A; allantois; W, Wolfan (mesonephric) duct; TG, tail gut; CM, cloacal membrane. Arrows point to the cranial and caudal borders of the CM. Large arrow points to the shallow urorectal fold.

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Figure 3 SEM photograph of the cloaca of a 14-day-old rat embryo. (A) Lateral view of the cloaca. A lateral ridge that would divide the dorsal and ventral cloacas is not seen (arrow). HG, hindgut; A, allantois, C, cloaca. (B) Ventral view of the cloaca (schematic drawing after SEM photograph; [C]). Signs of fusion of the lateral wall components are missing. The shape of the lower tip of the urorectal fold (small arrows) is evidence against fusion from cranial to caudal, W, left and right orices of Wolfan ducts; DC, dorsal (anorectal) part of the cloaca.

border of the body-stalk to the tail, where the tail gut enters the cloaca. At this stage, the cloaca has the shape of a triangle standing on its top. A genital tubercle is missing. In the following stages, the cloacal shape starts to change. This is caused by the ventral growth of the genital tubercle, a process that can be traced easily in a 14-day-old rat embryo (Figure 1B). This growth results in 2 processes: (1) a remarkable outgrowth of the cloaca into a ventral direction and (2) a rectangular displacement of the CM (Figure 1A-C), which swings down from a vertical to a horizontal position. The septum in normal cloacal development In a 12.5-day-old embryo (Figure 2), a tiny depression can be noted between the diverticulum of the urachus and the rectum. This fold is the rst indication of the so-called urogenital septum. Using the junction between the mesonephric duct and the cloaca as a marker, the descent of this fold can be discerned with ease (Figure 1). This is in contradiction to Van der Puttes10 observations. To see directly what happens during this so-called process of septation, we sagitally opened cloacas of 13-dayold embryos to inspect the cloacas from inside. However, lateral cloacal ridges or signs of fusion of lateral cloacal wall components were absent (Figure 3).

The fusion of the urorectal fold with the cloacal membrane in normal cloacal development In our studies we noted a disintegration of the CM in the area where the tip of the urorectal fold meets the CM (Figure 4). The region of the future anal orice It is interesting to note that, in the period of ventral cloacal shifting (between day 11 and day 15), the dorsal part of the CM and the dorsal cloaca always remains in close contact with the tail region. This region, which carries the anlage of the future anal orice, is the xed point in cloacal development (Figure 1).

Normal cloacal development (conclusions)

Nomenclature
It must be kept in mind that the term cloaca is used to describe not only a transitional organ system in human embryos but also a congenital anomaly and a normal organ in birds. This can lead to the false conclusion that the morphology of these 3 entities is similar. This is not

Figure 4 SEM photographs of the cloacas of 16-day-old rat embryos. (A) Lateral view of the cloaca after microdissection. The mesenchyme has been removed. The urorectal fold (URF) has nearly reached the cloacal membrane (CM). (B) In this slightly older embryo, the tip of the URF has reached the level of the CM. Local disintegration of the CM is obvious. U, urethra; R, rectum AO, anal opening; CE, cloacal epithelium.

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the case. Despite the same name, embryonic cloacas are completely different morphologically from cloacas in females with ARM and in birds. The main difference is the presence/absence of the area of the future anal opening. In embryonic cloacas, the future anal region is always present, whereas the anus is always missing in the human malformation that we call cloacas. This confusion in the terminology is the result of 2 outdated theories: Haeckels1 biogenetic law and the theory of the malformation as a frozen stage of normal embryology (Hemmungsmibildung2).

Conclusions from SEM studies

Our SEM studies clearly indicate that the subdivision of the cloaca is not the result of a process of fusion of lateral cloacal wall components.4 In our opinion, the importance of the process of septation has been overestimated in the past. According to our results, the normal development of the hindgut depends primarily on the normal formation of the CM. In all normal embryos, we could identify the region of the future anal orice in the dorsal part of the CM close to the tail groove. This observation makes the theory of a migration of the rectal opening to the perineum or a shift of the dorsal cloaca obsolete. Furthermore, it is obvious from our SEM observations that the embryonic cloaca never passes through a stage that is similar to any form of ARM in neonates, including the so-called cloacas in females. As mentioned previously, the importance of the subdivision of the embryonic cloaca has been overestimated. The most impressive feature in most cases of ARM is the missing anus, which seems to enter the urogenital tract as an ectopic rectal opening or is simply misplaced ventrally into the perineum. Obviously, this misplacement cannot be explained by a faulty septation alone because this would result in a persistent embryonic cloaca with the area of the future anal orice still in its place. According to our ndings, the downgrowth of the urorectal septum is the result of normal cloacal development, not its cause. A fusion of the urorectal fold with the CM could not be observed. When the fold comes in contact with the CM, it disintegrates locally.
Figure 5 Histologic section of the pelvic organs of an SD mouse (newborn). This newborn presents the features of an ARM with recto-urethral stula (F) and a blind ending rectal pouch (RP). U, urethra.

the rectum and the urogenital system, causing a spectrum of anorectal and urogenital anomalies.9 Recently, we analyzed the spectrum of anorectal anomalies in this model.37 The pathologic-anatomic ndings in the heterozygous (SD/ ) SD mouse group (Figure 5) were identical to those described earlier in pigs10 and humans.32,38 Because the percentage of abnormal animals per litter is high and breeding of SD mice is simple and inexpensive, we believe that the SD mouse model is ideal for studying the embryologic background of disturbed cloacal development.

Recent studies in SD mouse embryos

The SD mice used for this study originally were received from Philip Harris Biological, Ltd, England, in 1985, and were bred continuously in our facility until 1999 in accordance with German federal and local regulations. A total of 80 abnormal SD mouse embryos were identied easily by their shortened or crooked tails. In several of these, the genitals were abnormal as well (Figure 6). After microdissection, typical morphologic changes could be observed when abnormal and normal cloacas were compared (Figures 7 and 8). In all abnormal cloacas, we found the following: 1. An unusual shape of the cloaca. The dorsal cloaca was always missing. 2. The CM was too short. In all cases the dorsal part of the CM was absent. 3. An abnormal junction between the proximal hindgut and the cloaca.

Abnormal cloacal development

Until recently,35 the embryology of abnormal hindgut development was generally a matter of speculation. Progress in this eld has been hampered by lack of appropriate animal models that would permit systematic embryological studies in a sufcient series of malformed embryos. In 1940, a mutant of the normal house mouse, the SD-mutant, had been described by Dunn et al.36 These mice, rst bred by Danforth,8 prominently feature a short tail and therefore also are known as Danforths short tail mice.9 However, the SD gene inuences not only the axial skeleton but also

Recent advances in the studies of the embryology of ARM

As already mentioned, many animal models for ARM have been developed recently and were used for embryologic

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Figure 6 Abnormal SD mouse embryo. Note the crippled tail (T) and the hypoplastic genital tubercle (GT).

studies. Depending on the nature of the model, the following observations were made: 1. After feeding of 60 mg/kg etretinate (a long-acting synthetic retinoid) to pregnant mice on day 9, Kubota et al19 observed a specic pattern of cell proliferation and apoptosis in treated and untreated embryos on day 11 and 12. Although in control embryos proliferation was noted

Figure 8 SEM photograph of a normal (A) and an abnormal (B) 14-day-old SD mouse embryo. The ndings are identical to those in Figure 7. Note that the dorsal cloaca (DC) is missing.

in the area of the CM, proliferation was missing in age-matched embryos after etretinate ingestion. However, these embryos showed notable apoptosis in the dorsal area of the tail at the same time point. Interestingly, the differences were not so obvious in 12 day old embryos. 2. Another teratogen was used by Qi and coworkers25 in their study of the embryology of ARM. They fed pregnant rats with 1% ethylenthiourea (125 mg/kg) on gestational day 10. Embryos were harvested at day 13, 14, 15, and 16. In their study they veried most of our morphologic ndings in abnormal embryos. However, disagreement exists on whether the urorectal septum fuses with the CM or not. The appearance of the abnormal short CM and missing cloaca is explained by these authors by a combination of tail gut persistence and overwhelming apoptosis in the dorsal part of the cloaca. Interestingly, this apoptosis had not been observed by Kubota et al.19 Another genetic model of ARM has been presented by Mo et al.29 They studied mutant mice with various defects in the sonic hedgehog (shh) signaling pathway. Three mutants were studied: Shh null-mutant mice which showed

Figure 7 Schematic drawings of a normal (A) and an abnormal (B) cloaca. In the abnormal embryo, the cloacal membrane (CM) is too short (arrow). The cloacal membrane does not extend to the region of the tail groove (gray area). The dorsal cloaca is missing. In the normal embryo (A), the cloacal membrane is of normal length and extends to the region of the tail groove (gray area).

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4. Kluth D, Hillen M, Lambrecht W. The principles of normal and abnormal hindgut development. J Pediatr Surg 1995;30:1143-7. 5. Kluth D, Petersen C, Zimmermann HJ. The developmental anatomy of congenital diaphragmatic hernia. Pediatr Surg Int 1987;2:322-6. 6. Kluth D, Steding G, Seidl W. The embryology of foregut malformations. J Pediatr Surg 1987;22:389-93. 7. Kluth D, Kaestner M, Tibboel D, et al. Rotation of the gut: Fact or fantasy? J Pediatr Surg 1995;30:448-53. 8. Danforth CH. Developmental anomalies in a special strain of mice. Am J Anat 1930;45:275-87. 9. Gluecksohn-Schoenheimer S. The morphological manifestation of a dominant mutation in mice affecting tail and urogenital system. Genetics 1943;28:341-8. 10. van der Putte SCJ. Normal and abnormal development of the anorectum. J Pediatr Surg 1986;21:434-40. 11. Lambrecht W, Lierse W. The internal sphincter in anorectal malformations: Investigations in newborn pigs. J Pediatr Surg 1987;22: 1160-5. 12. Ikebukuro K-I, Ohkawa H. Three-dimensional analysis of anorectal embryology. A new technique for microscopic study using computer graphics. Pediatr Surg Int 1994;9:2-7. 13. Thompson DJ, Molello JA, Strebing RJ, et al. Teratogenicity of Adriamycin and daunomycin in the rat and rabbit. Teratology 1978;17: 151-8. 14. Diez-Pardo JA, Baoquan Q, Navarro C, et al. A new rodent experimental model of esophageal atresia and tracheoesophageal stula: Preliminary report. J Pediatr Surg 1996;31:498-502. 15. Beasley SW, Diez Pardo J, Qi BQ, et al. The contribution of the Adriamycin-induced rat model of the VATER association to our understanding of congenital abnormalities and their embryogenesis. Pediatr Surg Int 2000;16:465-72. 16. Orford J, Manglick P, Cass DT, et al. Mechanisms for the development of esophageal atresia. J Pediatr Surg 2001;36:985-94. 17. Gillick J, Giles S, Bannigan S, et al. Midgut atresias result from abnormal development of the notochord in an Adriamycin rat model. J Pediatr Surg 2002;37:719-22. 18. Merei JM, Hutson JM. Embryogenesis of tracheoesophageal anomalies: A review. Pediatr Surg Int 2002;18:319-26. 19. Kubota Y, Shimotake T, Yanagihara J, et al. Development of anorectal malformations using etretinate. J Pediatr Surg 1998;33:127-9. 20. Liu Y, Sugiyama F, Yagami K, et al. Sharing of the same embryogenic pathway in anorectal malformations and anterior sacral myelomeningocele formation. Pediatr Surg Int 2003;19:152-6. 21. Bitoh Y, Shimotake T, Sasaki Y, et al. Development of the pelvic oor muscles of murine embryos with anorectal malformations. J Pediatr Surg 2002;37:224-7. 22. Hashimoto R, Nagaya M, Ishiguro Y, et al. Relationship of the stulas to the rectum and genitourinary tract in mouse fetuses with high anorectal malformations induced by all-trans retinoic acid. Pediatr Surg Int 2002;18:723-7. 23. Sasaki Y, Iwai N, Tsuda T, et al. Sonic hedgehog and bone morphogenetic protein 4 expressions in the hindgut region of murine embryos with anorectal malformations. J Pediatr Surg 2004;39:170-3. 24. Arana J, Villanueva A, Guarch R, et al. Anorectal atresia. An experimental model in the rat. Eur J Pediatr Surg 2001;11:192-5. 25. Qi BQ, Beasley SW, Frizelle FA. Clarication of the processes that lead to anorectal malformations in the ETU-induced rat model of imperforate anus. J Pediatr Surg 2002;37:1305-12. 26. Yuan ZW, Lui VC, Tam PK. Decient motor innervation of the sphincter mechanism in fetal rats with anorectal malformation: A quantitative study by uorogold retrograde tracing. J Pediatr Surg 2003;38:1383-8. 27. Bai Y, Chen H, Yuan ZW, et al. Normal and abnormal embryonic development of the anorectum in rats. J Pediatr Surg 2004;39:58790. 28. Kim J, Kim P, Hui CC, the VACTERL Association: Lessons from the sonic hedgehog pathway. Clin Genet 2001;59:306-15.

persistent cloacas, Gli2 mutant mice which demonstrated the classic form of ARMs and Gli3 mutants with anal stenosis. They conclude from their observations that Shh signaling is essential for the normal development of the hindgut. Interestingly, the morphology of Gli2 mutant mice embryos resembles that of heterozygous SD-mice embryos, whereas Shh-null mutant mice embryos had morphologic similarities with homozygous SD mice embryos.

Conclusions
Traditionally, the normal differentiation of the cloaca into the dorsal anorectum and the ventral urogenital tract is attributable to the proper process of septation by the socalled urorectal septum.38 However, when we used SEM in our study, we noted that neither lateral ridges nor signs of fusing lateral wall components could be discerned. Therefore, clear proof of this process of septation is still missing. It is more likely that a normal-looking septum is the result of normal cloacal development rather than its cause. Our study on the normal embryology of the hindgut clearly demonstrates that the area of the future anal orice is formed in an early phase of development and forms a xed point in cloacal and hindgut development. In contrast to this, it appears that investigators still assume that a shift of the rectum (caudal migration) or a shift of the caudal cloaca to the tail groove10,33,34 takes place, which is necessary to establish the anorectal canal. Our results clearly indicate that this assumption is obsolete. It is essential to note that in all abnormal SD mouse embryos the dorsal CM and the dorsal cloaca were missing. Both structures are essential for the normal establishment of the anal orice and the lower rectum. Therefore, it is not surprising that a defective cloacal anlage results in a missing or misplaced anal orice and an abnormal communication between the rectum and the ventral urogenital tract. Recent studies by Mo and co-workers29 demonstrated the importance of the Gli2 transcription factor for the normal development of the hindgut. Our results further indicate that the abnormal cloacas found in our SD-mice are not part of the normal spectrum of cloacal development that we observed in normal rat embryos. This means that the cloaca of a normal embryo will never result in ARM. Therefore, the term persistent cloaca for the human anomaly is a misnomer.

References
1. Haeckel E, cited in Starck D. Embryologie (ed 3). Stuttgart, Germany: Thieme, 1975. 2. Gilbert SF. Developmental Biology (ed 7) Chapter 23. Sunderland, MA: Sinauer Associates, 2003. 3. Schwalbe E. The morphology of the malformations in human and animals. Part 1: General Teratology. Jena, Germany: Gustav Fischer, 1906 :143-4.

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29. Mo R, Kim JH, Zhang J, et al. Anorectal malformations caused by defects in sonic hedgehog signaling. Am J Pathol 2001;159:765-74. 30. Tourneux F. On the early development of the cloaca, the genital tubercles and the anus in sheep embryos, including some remarks on the development of the prostatic glands. J Anat Physiol 1888;24:50317. 31. Retterer E. Sur 1origin et de 1evolution de la region Ano-genitale des mammiferes. J Anat Physiol 1890;26:126-210. 32. Stephens FD. Congenital Malformations of the Rectum, Anus, and Genitourinary Tract. Edinburgh, UK: Livingstone, 1963. 33. Bill AH, Johnson RJ. Failure of migration of the rectal opening as the cause for most cases of imperforate anus. Surg Gynecol Obstet 1958; 106:643-51.

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34. Gans SL, Friedman NB. Some new concepts in the embryology, anatomy, physiology, and surgical correction of imperforate anus. West J Surg Obstet Gynecol 1961;63:34-7. 35. Hutson JM, van der Putte SCJ, Penington E, et al. The embryology of anorectal malformations. In Holschneider AM, Hutson JM, eds. Anorectal Malformations in Children. Berlin, Heidelberg: Springer, 2006:49-63. 36. Dunn LC, Gluecksohn-Schoenheimer S, Bryson V. A new mutation in the mouse affecting spinal column and urogenital system. J Hered 1940;31:343-8. 37. Kluth D, Lambrecht W, Reich P, et al. SD-miceAn animal model for complex anorectal malformations. Eur J Pediatr Surg 1991;1:183-8. 38. Gray SW, Skandalakis JE. Embryology for Surgeons. Philadelphia, PA: W. B. Saunders, 1972: 187-216.