Embryology of Anorectal Malformations

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

mbryology of anorectal malformations

ietrich Kluth, MD, PhD

rom the Department of Pediatric Surgery, University Hospital, Leipzig University, Leipzig, Germany.

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 finally leadto abnormal hindgut development are better known than in the past: (1) the process of maldevelopmentstarts 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 fistula. In the past, an impaired process of septation was believed to be themain cause of abnormal hindgut development. In contrast to this, our results indicate that the devel-opment of the septum is more passive than active. Furthermore, the results of our studies in normal andabnormal development indicate that (1) the embryonic cloaca never passes through a stage that issimilar to any form of anorectal malformation in neonates, including the so-called “cloacas” in femaleembryos, and (2) to explain abnormal development, studies in abnormal embryos are mandatory.© 2010 Elsevier Inc. All rights reserved.

KEYWORDSNormal anorectaldevelopment;Embryology;Abnormaldevelopment;Animal models;Cloaca;Hindgut

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Despite many efforts, the embryology of numerous con-enital anomalies in humans is still a matter of speculationecause of the following:

. A shortage of study material (both normal and abnormalembryos).

. Various technical problems (difficulties in the interpre-tation of serial sections, shortage of explanatory three-dimensional reconstructions).

. Misconceptions and/or outdated theories concerning nor-mal and abnormal embryology.

Fortunately, many animal models are known today thatllow advanced embryologic studies in various embryologicelds. Especially for the studies of anorectal malformationsARMs), several animal models exist. However, appropriatend illustrative descriptions in various fields of embryologyre still lacking, which explains why today many typical

Supported in part by Deutsche Forschungsgemeinschaft, Grant NumberL 596/1, Hamburg Werner-Otto-Stiftung.

Address reprint requests and correspondence: Dietric Kluth, MD,hD, Department of Pediatric Surgery, University Hospital, Leipzig Uni-ersity, Liebigstraß 18, 04103 Leipzig, Germany.

fE-mail address: [email protected].

055-8586/$ -see front matter © 2010 Elsevier Inc. All rights reserved.oi:10.1053/j.sempedsurg.2010.03.005

alformations are still not sufficiently explained. Pediatricurgeons are still confused when they are confronted withhe embryologic background of normal and abnormal de-elopment.

For misconceptions and/or outdated theories, Haeck-l’s “biogenetic law”1 is one example. According to thisheory, the human embryo recapitulates in its individualevelopment (ontogeny) the morphology observed in allife forms (phylogeny). This means that during its devel-pment, an advanced species is seen to pass throughtages represented by adult organisms of more primitivepecies.2 This theory has still an impact on the nomen-lature of embryonic organs. This explains why humanmbryos have “cloacas” like adult birds and “branchial”lefts like adult fish.

Another very popular misconception is the theory thatalformations actually represent “frozen” stages of normal

mbryology (“Hemmungsmißbildung”).3 As a result, ournderstanding of normal embryology stems rather from thenterpretations of observed malformations than from propermbryologic observations. The theory of the “rotation of theut” as a step in normal development is a perfect example
or this misconception.
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202 Seminars in Pediatric Surgery, Vol 19, No 3, August 2010

The purpose of this work is to illustrate what we haveearned from our studies in normal and abnormal hindgutevelopment performed in our laboratory. We used scan-ing electron microscopy (SEM) to illustrate our findings.4

n our experience, SEM allows the documentation of 3-di-ensional embryonic structures in superior detail.5-7

he etiology of anorectal malformations

he etiology of ARMs is still unclear. Most researchersssume that its etiology is multifactorial. However, recentlynumber of animal models has been deployed to study theackground of ARM. In these models genetic as well asnvironmental factors were identified.

. The SD-mouse model: these mice, first bred by Dan-forth,8 prominently feature a short tail and therefore alsoare known as “Danforth’s short tail mice.”9

. The pig model: another famous model for abnormalhindgut development is the pig.10-12

. The adriamycin model: in 1978, Thompson et al13 usedadriamycin to induce various anomalies in high percent-ages in rat fetuses. Observed were esophageal atresiaswith tracheoesophageal fistulas and ARMs amongst oth-ers. This model of the “VACTERL association” has beenstudied in detail by numerous research groups.14-18

Other substances that have proven to be useful in induc-ng ARM (mainly in rats) are etretinate,19,20 all-trans reti-oic acid,21-23 and ethylenethiourea.24-27

Recently, Kim et al28 and Mo et al29 studied malforma-ions in Gli mutant mice and found a spectrum of abnor-alities similar to those in the VACTERL association. They

ould demonstrate that a modulation of the defective gene

igure 1 Schematic drawing of normal cloacal development in rmbryo; and (C) 15-day embryo. Note the movement of the cloacals caused by the ventral outgrowth of the genital tubercle and theart of the cloacal membrane (gray dots) is the area of the future as the fixed point in development of the cloaca. HG, hindgut; C
rogenitalis; W, Wolffian (mesonephric) duct; U, ureter.
esulted in a spectrum of ARMs as seen in humans. Inrinciple, all these models are sufficient to study the em-ryology of abnormal hindgut development. Interestingly,ll animal models presented with a similar spectrum ofbnormalities.

ormal embryology of the hindgut

he normal embryology of the hindgut always has been aatter of debate because observations made in normal em-

ryos should not only explain the normal embryology butlso its abnormal counterpart. As a result, the explanation oformal embryology was always done with abnormal devel-pment in mind. Two major theories exist to explain theifferentiation of the hindgut into the urogenital (ventral)nd anorectal (dorsal) part:

. The theory of the septation of the cloaca; and

. The theory of the migration of the rectum.

The latter had been modified by van der Putte10 in 1986.nother controversy exists of whether the urorectal septum

uses with the cloacal membrane (CM) in normal develop-ent or not.

arly development of the hindgut

he “anorectal septum” of the hindgutIn very young embryos, the hindgut is a simple structure

Figure 1). Cranially, it is in continuity with the midgut;audally, it is in direct contact with the ectoderm, thusorming the “cloacal membrane.” When developmentrogresses, the caudal part of the hindgut, the “cloaca,”

awn after SEM photographs). (A) A 12.5-day embryo; (B) 14-dayrane (CM) from a vertical to a horizontal position. This movement. Note the descent of the urorectal fold (short arrows). The dorsalening. Arrows with asterisk (*) point to the tail groove. This areaoacal membrane; C, cloaca; TG, tail gut; A, allantois; S, sinus

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203Kluth Embryology of Anorectal Malformations

ifferentiates into 2 separate organ systems—the urogenitalract and the anorectal tract.

Since the work of Tourneux30 and Retterer31 at the endf the 19th century, it has been generally accepted that theormal development of these tracts depends on the properubdivision of the cloaca by a septum, the so-called “uro-ectal septum.” According to this theory, abnormal septalevelopment always should result in abnormal cloacal de-elopment. However, there is no agreement among inves-igators about the nature of this septum and the way itevelops. Although Tourneux30 thought that the septumoves down from cranial to caudal “like a French curtain,”etterer31 speculated that lateral folds or ridges appear in

he lumen of the cloaca. These ridges should fuse and thusorm the septum, beginning cranial and ending caudal at theevel of the CM. In the past, numerous investigators sup-orted one of these theories. Stephens32 combined bothheories, believing that this could best explain the variousorms of ARMs. He claimed that the cranial part of theeptum should grow downward as explained by Tourneux,hereas in the caudal part lateral ridges should fuse to form

he septum in this area. In 1986, van der Putte10 denied theajor role of the urorectal septum in the process of “cloa-

al” differentiation.

he “migration” of the rectumStudying the morphology of ARM in human newborns,

ill and Johnson33 and later Gans and Friedman34 statedhat in most forms of ARM the fistula may present anectopic” anal opening. They concluded from these obser-ations that the rectum actually “migrates” during normalevelopment, from a rather high position to the normal areaf the anal opening. If this “process of migration” stopsefore the anus has reached its definitive position in the areaf the perineum, an ectopic anal canal would result. Al-hough this speculation is rather attractive, neither thesenvestigators nor other researchers were able to show anymbryologic evidence of this “migration.”

he “shift of the dorsal cloaca”In 1986, van der Putte10 modified the theory of a “rectal”

r “anal” migration. While studying normal and abnormalig embryos, he speculated that a “shift” of the dorsal cloacaakes place. This shift should bring the dorsal cloaca downo the area of the tail groove, thus establishing here theuture anal opening.

tudies in the normal embryology of the hindgutauthor’s studies)

n 1995, we studied hindgut development in a series oftaged rat embryos between the 10th and the 15th gesta-ional day (comparable with human embryos between thehird and 7th week of gestation and rat embryos between the1th and 16th gestational day)4 by using SEM. A total of
45 embryos were analyzed in this study. u
The SEM was used because of the following advantages:

. Serial sectioning of embryos and time-consuming 3-di-mensional reconstructions are not necessary.

. The embryo can be studied in all 3 dimensions “on-line.”

. The images and photographs are of superior quality.

The essential findings of our study are summarized inigure 1.

In contrast to earlier reports, we found that (1) a septationf the cloaca by fusion of the lateral folds does not takelace and (2) a migration of the anal opening or a shifting ofhe dorsal cloaca cannot be observed.

he early “cloacal” developmentThe starting point of this series is the “cloaca” in a

2.5-day-old rat embryo (Figure 2). At this stage, all fea-ures of a typical “cloaca” are present: the hindgut (HG)nters the cloaca (c) from dorsocranial whereas the allantoisa) (the forerunner of the bladder) can be identified as aranioventral diverticulum. Between this diverticulum andhe hindgut, the urorectal fold (arrow) can be seen. This foldarks the cranial border of the undifferentiated hindgut, the

o-called “cloaca.”The mesonephric duct (Wolffian duct; W) enters the

loaca in its cranial part but in a relatively dorsal position.audally, the cloaca continues directly into the tailgut (TG).he CM extends in a slight concave curve from the caudal

igure 2 SEM photograph of the cloaca of a 12.5-day-old ratmbryo. Lateral view of the cloaca after microdissection. Theesenchyme has been removed. See text for details. C, cloaca;G; hindgut; A; allantois; W, Wolffian (mesonephric) duct; TG,

ail gut; CM, cloacal membrane. Arrows point to the cranial andaudal borders of the CM. Large arrow points to the shallow
rorectal fold.

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order of the body-stalk to the tail, where the tail gut entershe cloaca. At this stage, the cloaca has the shape of ariangle standing on its top. A genital tubercle is missing.

In the following stages, the cloacal shape starts tohange. This is caused by the ventral growth of the genitalubercle, a process that can be traced easily in a 14-day-oldat embryo (Figure 1B). This growth results in 2 processes:1) a remarkable outgrowth of the cloaca into a ventralirection and (2) a rectangular displacement of the CMFigure 1A-C), which swings down from a vertical to aorizontal position.

he “septum” in normal cloacal developmentIn a 12.5-day-old embryo (Figure 2), a tiny depression

an be noted between the diverticulum of the urachus andhe rectum. This fold is the first indication of the so-calledrogenital “septum.” Using the junction between the meso-ephric duct and the cloaca as a marker, the descent of thisold can be discerned with ease (Figure 1). This is inontradiction to Van der Puttes10 observations.

To see directly what happens during this so-called pro-ess of “septation,” we sagitally opened cloacas of 13-day-ld embryos to inspect the cloacas from inside. However,ateral cloacal ridges or signs of fusion of lateral cloacalall components were absent (Figure 3).

igure 3 SEM photograph of the cloaca of a 14-day-old rat embryond ventral cloacas is not seen (arrow). HG, hindgut; A, allantois,hotograph; [C]). Signs of fusion of the lateral wall components arevidence against fusion from cranial to caudal, W, left and right orifi

igure 4 SEM photographs of the cloacas of 16-day-old rat embryas been removed. The urorectal fold (URF) has nearly reached the c
as reached the level of the CM. Local disintegration of the CM is obvious. U,
he fusion of the urorectal fold with the cloacalembrane in normal cloacal developmentIn our studies we noted a disintegration of the CM in the

rea where the tip of the urorectal fold meets the CMFigure 4).

he region of the future anal orificeIt is interesting to note that, in the period of ventral

loacal shifting (between day 11 and day 15), the dorsal partf the CM and the dorsal cloaca always remains in closeontact with the tail region. This region, which carries thenlage of the future anal orifice, is the fixed point in cloacalevelopment (Figure 1).

ormal cloacal development (conclusions)

omenclature

t must be kept in mind that the term “cloaca” is used toescribe not only a transitional organ system in humanmbryos but also a congenital anomaly and a normalrgan in birds. This can lead to the false conclusion thathe morphology of these 3 entities is similar. This is not

ateral view of the cloaca. A lateral ridge that would divide the dorsalca. (B) Ventral view of the cloaca (schematic drawing after SEMg. The shape of the lower tip of the urorectal fold (small arrows) isWolffian ducts; DC, dorsal (anorectal) part of the cloaca.

) Lateral view of the cloaca after microdissection. The mesenchymemembrane (CM). (B) In this slightly older embryo, the tip of the URF

. (A) LC, cloamissin

os. (Aloacal

urethra; R, rectum AO, anal opening; CE, cloacal epithelium.

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he case. Despite the same name, embryonic cloacas areompletely different morphologically from cloacas inemales with ARM and in birds. The main difference ishe presence/absence of the area of the future anal open-ng. In embryonic cloacas, the future anal region is al-ays present, whereas the anus is always missing in theuman malformation that we call “cloacas.” This confu-ion in the terminology is the result of 2 outdated theo-ies: Haeckel’s1 “biogenetic law” and the theory of thealformation as a “frozen” stage of normal embryology

“Hemmungsmißbildung”2).

onclusions from SEM studies

ur SEM studies clearly indicate that the subdivision ofhe cloaca is not the result of a process of fusion of lateralloacal wall components.4 In our opinion, the importancef the process of septation has been overestimated in theast. According to our results, the normal development ofhe hindgut depends primarily on the normal formation ofhe CM. In all normal embryos, we could identify theegion of the future anal orifice in the dorsal part of theM close to the tail groove. This observation makes the

heory of a “migration” of the rectal opening to theerineum or a “shift” of the dorsal cloaca obsolete. Fur-hermore, it is obvious from our SEM observations thathe embryonic cloaca never passes through a stage that isimilar to any form of ARM in neonates, including theo-called “cloacas” in females.

As mentioned previously, the importance of the subdi-ision of the embryonic cloaca has been overestimated. Theost impressive feature in most cases of ARM is the miss-

ng anus, which seems to enter the urogenital tract as anectopic” rectal opening or is simply misplaced ventrallynto the perineum. Obviously, this misplacement cannot bexplained by a faulty septation alone because this wouldesult in a persistent embryonic cloaca with the area of theuture anal orifice still in its place. According to our find-ngs, the downgrowth of the urorectal septum is the result oformal cloacal development, not its cause. A fusion of therorectal fold with the CM could not be observed. When theold comes in contact with the CM, it disintegrates locally.

bnormal cloacal development

ntil recently,35 the embryology of abnormal hindgut de-elopment was generally a matter of speculation. Progressn this field has been hampered by lack of appropriatenimal models that would permit systematic embryologicaltudies in a sufficient series of malformed embryos. In 1940,mutant of the normal house mouse, the SD-mutant, had

een described by Dunn et al.36 These mice, first bred byanforth,8 prominently feature a short tail and therefore

lso are known as “Danforth’s short tail mice.”9 However,
he SD gene influences not only the axial skeleton but also b
he rectum and the urogenital system, causing a spectrum ofnorectal and urogenital anomalies.9 Recently, we analyzedhe spectrum of anorectal anomalies in this model.37 Theathologic-anatomic findings in the heterozygous (SD/�)D mouse group (Figure 5) were identical to those de-cribed earlier in pigs10 and humans.32,38 Because the per-entage of abnormal animals per litter is high and breedingf SD mice is simple and inexpensive, we believe that theD mouse model is ideal for studying the embryologicackground of disturbed cloacal development.

ecent studies in SD mouse embryos

he SD mice used for this study originally were receivedrom Philip Harris Biological, Ltd, England, in 1985, andere bred continuously in our facility until 1999 in ac-

ordance with German federal and local regulations. Aotal of 80 abnormal SD mouse embryos were identifiedasily by their shortened or crooked tails. In several ofhese, the genitals were abnormal as well (Figure 6). Aftericrodissection, typical morphologic changes could be ob-

erved when abnormal and normal cloacas were comparedFigures 7 and 8). In all abnormal cloacas, we found theollowing:

. An unusual shape of the cloaca. The dorsal cloaca wasalways missing.

. The CM was too short. In all cases the dorsal part of theCM was absent.

. An abnormal junction between the proximal hindgut andthe cloaca.

ecent advances in the studies of the embryologyf ARM

s already mentioned, many animal models for ARM have

igure 5 Histologic section of the pelvic organs of an SD mousenewborn). This newborn presents the features of an ARM withecto-urethral fistula (F) and a blind ending rectal pouch (RP). U,rethra.

een developed recently and were used for embryologic

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tudies. Depending on the nature of the model, the follow-ng observations were made:

. After feeding of 60 mg/kg etretinate (a long-acting syn-thetic retinoid) to pregnant mice on day 9, Kubota et al19

observed a specific pattern of cell proliferation and ap-optosis in treated and untreated embryos on day 11 and12. Although in control embryos proliferation was noted

igure 6 Abnormal SD mouse embryo. Note the crippled tailT) and the hypoplastic genital tubercle (GT).

igure 7 Schematic drawings of a normal (A) and an abnormalB) cloaca. In the abnormal embryo, the cloacal membrane (CM)s too short (arrow). The cloacal membrane does not extend to theegion of the tail groove (gray area). The dorsal cloaca is missing.n the normal embryo (A), the cloacal membrane is of normal

tength and extends to the region of the tail groove (gray area).

in the area of the CM, proliferation was missing inage-matched embryos after etretinate ingestion. How-ever, these embryos showed notable apoptosis in thedorsal area of the tail at the same time point. Interest-ingly, the differences were not so obvious in 12 day oldembryos.

. Another teratogen was used by Qi and coworkers25 intheir study of the embryology of ARM. They fed preg-nant rats with 1% ethylenthiourea (125 mg/kg) on ges-tational day 10. Embryos were harvested at day 13, 14,15, and 16. In their study they verified most of ourmorphologic findings in abnormal embryos. However,disagreement exists on whether the urorectal septumfuses with the CM or not.

The appearance of the abnormal short CM and missingloaca is explained by these authors by a combination of tailut persistence and overwhelming apoptosis in the dorsalart of the cloaca. Interestingly, this apoptosis had not beenbserved by Kubota et al.19

Another genetic model of ARM has been presented byo et al.29 They studied mutant mice with various defects

n the sonic hedgehog (shh) signaling pathway. Three mu-

igure 8 SEM photograph of a normal (A) and an abnormal (B)4-day-old SD mouse embryo. The findings are identical to thosen Figure 7. Note that the dorsal cloaca (DC) is missing.

ants were studied: Shh null-mutant mice which showed

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persistent cloacas,” Gli2 mutant mice which demonstratedhe “classic” form of ARMs and Gli3 mutants with analtenosis. They conclude from their observations that Shhignaling is essential for the normal development of theindgut. Interestingly, the morphology of Gli2 mutant micembryos resembles that of heterozygous SD-mice embryos,hereas Shh-null mutant mice embryos had morphologic

imilarities with homozygous SD mice embryos.

onclusions

raditionally, the normal differentiation of the cloaca intohe dorsal anorectum and the ventral urogenital tract isttributable to the proper process of septation by the so-alled urorectal septum.38 However, when we used SEM inur study, we noted that neither lateral ridges nor signs ofusing lateral wall components could be discerned. There-ore, clear proof of this process of septation is still missing.t is more likely that a normal-looking septum is the resultf normal cloacal development rather than its cause.

Our study on the normal embryology of the hindgutlearly demonstrates that the area of the future anal orifices formed in an early phase of development and forms axed point in cloacal and hindgut development. In contrast

o this, it appears that investigators still assume that a “shift”f the rectum (“caudal migration”) or a shift of the caudalloaca to the tail groove10,33,34 takes place, which is neces-ary to establish the anorectal canal. Our results clearlyndicate that this assumption is obsolete.

It is essential to note that in all abnormal SD mousembryos the dorsal CM and the dorsal cloaca were missing.oth structures are essential for the normal establishment of

he anal orifice and the lower rectum. Therefore, it is noturprising that a defective cloacal anlage results in a missingr misplaced anal orifice and an abnormal communicationetween the rectum and the ventral urogenital tract. Recenttudies by Mo and co-workers29 demonstrated the impor-ance of the Gli2 transcription factor for the normal devel-pment of the hindgut.

Our results further indicate that the abnormal cloacasound in our SD-mice are not part of the normal spectrum ofloacal development that we observed in normal rat em-ryos. This means that the cloaca of a normal embryo willever result in ARM. Therefore, the term “persistent clo-ca” for the human anomaly is a misnomer.

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Embryology of Anorectal Malformations