Spatiotemporal Distribution of Apoptosis During Normal Cloacal Development in Mice

Home , Cloacal membrane, Cloaca (embryology), Urorectal septum

THE ANATOMICAL RECORD PART A 279A:761–767 (2004)

CHIHARU SASAKI, KUMIKO YAMAGUCHI, AND KEIICHI AKITA* Unit of Clinical Anatomy, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

ABSTRACT To understand normal cloacal developmental processes, serial sagittal sections of mouse embryos were made every 6 hrs from embryonic day 11.5 (E11.5) to E13.5. During cloacal development to form the urogenital sinus and anorectal canal, fusion of the urorectal septum with the cloacal membrane was not observed, and the ventral and dorsal parts of the cloaca were continuously connected by the canal until disappearance of the cloacal membrane to open the vestibule formed by the urogenital sinus and anorectal canal to the outside at E13.5. Ventral shifting of the dorsal part of the cloaca was observed until E12.5. The dorsal part was transformed in accordance with ventral shifting. In addition, apoptosis was seen to occur around the dorsal part. However, from E12.25, apoptotic cells are observed in a linear arrangement in the urorectal septum just ventral to the peritoneal cavity. Interestingly, extension of this line reaches the area of the cloacal membrane disintegrated by apoptosis. The present ﬁndings suggest that in the early stages (until E12.0), distribution of apoptosis in mesenchyme around the dorsal part of the cloaca might be strongly related to the transformation and ventral shifting of this part. Conversely, the apoptosis pattern in urorectal septum mesenchyme in later stages (from E12.0) might be involved in transformation of the urorectal septum and disintegration of the cloacal membrane. © 2004 Wiley-Liss, Inc.

Key words: normal cloacal development; mouse development; apoptosis; TUNEL method; genital tubercle

Despite a long history of embryological research, the part of the cloaca and transformations of the distal part of developmental processes of the anorectal canal remain the hindgut were also observed. contentious. The most debated point is whether fusion of Such dramatic changes in the morphology and configu- the urorectal septum with the cloacal membrane occurs in ration of embryonic structures in the cloacal region are normal development (Keibel, 1895; Pohlman, 1911; considered to be the result of embryonic cell differentia- Politzer, 1931; De Vries and Friedland, 1974a, 1974b; Van tion, cell proliferation, and apoptosis (programmed cell der Putte and Neeteson, 1983; Van der Putte, 1986; Ste- death). Cell death is known to play an important role in phens and Smith, 1988; Kluth et al., 1995; Miller and the formation of various embryonic organs and is recog- Briglin, 1996; Nievelstein et al., 1998; Kromer, 1999; Pai- nized as an important contributor to various areas inte- das et al., 1999; Qi et al., 2000a, 2000b, 2000c). In addi- gral to vertebrate development, such as limbs and digits tion, discussion has also centered around whether the (Saunders et al., 1962; Saunders and Fallon, 1967; Mori et cloacal membrane is divided into urogenital and anal membranes by the urorectal septum. To determine possible answers to these problems, we examined serial sagittal sections in mouse embryos from embryonic day 11.5 Grant sponsor: Grant-in-Aid for Scientific Research of the Min- istry and Education, Culture, Sports, Science and Technology; (E11.5) to E13.5 in detail, with particular focus on mor- Grant number: 13671639. phological changes in the dorsal part of the cloaca. Accord- *Correspondence to: Dr. Keiichi Akita, Unit of Clinical Anat- ing to the present findings, neither fusion of the urorectal omy, Graduate School, Tokyo Medical and Dental University, septum with the cloacal membrane nor division of the 1-5-45 Yushima Bunkyo Tokyo 113-8519, Japan. Fax: 81-3-5803- cloacal membrane occurred, as mentioned by Nievelstein 0116. E-mail: [email protected] et al. (1998) in humans. Disintegration of the cloacal mem- Received 5 February 2004; Accepted 4 May 2004 brane is observed in one region, forming a vestibule into DOI 10.1002/ar.a.20062 which the urogenital sinus and hindgut open. During clo- Published online 7 July 2004 in Wiley InterScience acal developmental processes, ventral shift of the dorsal (www.interscience.wiley.com).

© 2004 WILEY-LISS, INC. 762 SASAKI ET AL. al., 1995), heart (Pexieder, 1975), and tooth germs (Sasaki RESULTS et al., 2001). The present study also examined distribution Development and Growth of Genital Tubercle of apoptotic cells and bodies in the cloacal region. Distri- Affects Cloacal Development bution of apoptosis in the epithelial layers has already been reported by Qi et al. (2000b), and the present find- We examined normal development of the cloacal region ings are similar. However, spatial and temporal distribu- sequentially using serial sagittal sections of mouse em- tion of apoptosis in the mesenchyme remains unclear. bryos from E11.5 to E13.5 (Fig. 1A–J). In addition, sche- Computer-assisted three-dimensional reconstruction im- matic representation revealed development processes of ages were useful in understanding the spatial distribu- the cloacal region based on the present studies. Cloacal tions of apoptosis, and dramatic changes in the distribu- development started before E11.5, and the cloaca was tion of apoptosis in mesenchyme were noted at completely divided into the urogenital sinus and anorectal approximately E12.0. Before this stage, distribution is canal by E13.5. During these stages, the genital tubercle primarily around the dorsal cloaca, but is subsequently developed and grew substantially. Ventrocaudal out- found in the urorectal septum as a line that, when ex- growth of the genital tubercle caused dorsocaudal rotation tended, passes through the area of disintegration of the of the cloacal membrane. The cloaca shifted ventrocau- cloacal membrane. This spatiotemporal distribution of ap- dally according to genital tubercle development, and the optosis is deeply involved with the developmental pro- distal part of the hindgut also shifted. In the cloaca, the cesses of the cloaca, and possible roles of apoptosis in urorectal septum was observed in sagittal sections, and cloacal development are discussed herein. the septum divided the cloaca into ventral and dorsal parts. During development, the ventral part of the cloaca MATERIALS AND METHODS was expanded ventrally according to development of the Animals and Tissue Preparation for Light genital tubercle, whereas the dorsal part decreased in size Microscopy to become the distal part of the hindgut, which could Mature female ICR mice (SLC, Shizuoka, Japan) were represent a precursor to the anorectal canal. mated overnight with a male mouse. The morning of the Ventral and Dorsal Parts of Cloaca Are day on which a vaginal plug was found was designated as Continuously Connected E0.5. For sequential examination of normal development in the cloacal region, two pregnant mice were sacrificed As mentioned above, whether the urorectal septum every 6 hr from E11.5 to E13.5 (n ϭ 18). Handling of fuses with the cloacal membrane has long been debated. animals conformed to the guidelines for care and use of We examined serial sagittal sections of mouse embryos experimental animals as established by the Institutional during cloacal development to observe the border between Animal Care and Use Committee of Tokyo Medical and the ventral and dorsal parts of the cloaca. At E11.5 and Dental University (no. 10087). Embryos were fixed in 10% E11.75, no clear border between the two parts of the cloaca formalin, dehydrated, and embedded in paraffin. A total of was apparent, and the distal part of the hindgut and 52 embryos were serially sectioned in the sagittal plane at proximal part of the tailgut opened into the dorsal wall of 5 ␮m thickness. In two embryos at each stage, all sections the cloaca (Figs. 1A, B, F, G, K, and L and 2). From E12.0 were stained with hematoxylin and eosin (H&E). In all to E12.25, the urorectal septum was transformed accord- other embryos, sections were collected alternately, and ing to ventral shifting of the hindgut and tailgut (Figs. 1C, one subset was stained with H&E. H, and M and 3). The ventrocaudal part of the urorectal In order to confirm the presence of apoptotic cells and septum descended and expanded. The dorsal part of the bodies, nuclear DNA fragmentation of apoptotic cells in cloaca became a distal part of the hindgut, and the tailgut the other subset were labeled using TUNEL methods began to disappear. Outgrowth of the septum made a (Gavrieli et al., 1992) with an In Situ Cell Death Detection small canal between the two parts of the cloaca by E12.25. Kit (Roche Diagnostics, Tokyo, Japan). Peroxidase activity The ceiling of the canal was formed by epithelial layers of was visualized by immersion for 10 min in 0.02% diami- the tip of the urorectal septum, and the floor of the canal nobenzidine (DAB) in 0.05 mol/l Tris-HCl buffer (pH 7.4) was formed by the cloacal membrane. containing 0.01% H2O2. These sections were compared At E12.5 and E12.75, the dorsalmost part of the cloacal with adjacent H&E-stained sections to examine the dis- membrane started to disintegrate (Figs. 1D, I, and N and tribution of apoptotic cells and bodies and intensely he- 4). Disintegration occurred between the tip of the urorec- matoxylin-stained granules. tal septum and the dorsal end of the cloacal membrane. The position of the dorsal end of the cloacal membrane Three-Dimensional Reconstructions was similar to that of the junction of the perineal region Spatiotemporal distribution of pyknotic cells, apoptotic and tail bud until E12.25, but shifted ventrally thereafter. cells, and/or bodies was analyzed using computer-assisted The canal was still observed between the urogenital sinus three-dimensional reconstruction. Three-dimensional re- and distal part of the hindgut (Fig. 4E), but was so thin constructions were made from serial paraffin sections of that only one section from each embryo showed this struc- E11.5, E11.75, and E12.0 specimens stained with H&E. ture in the serial sagittal sections (Fig. 4B and D). Com- All sections were photographed and epithelial layers were plete fusion of the septum and cloacal membrane was not traced. Findings from sections with respect to the spatio- observed. The cloacal membrane disintegrated in the dis- temporal distribution of granular substances intensely tal end of the hindgut to form a small vestibule, and the stained with H&E were marked, and distinction was made canal and hindgut opened into this vestibule. between granular substance in the epithelial layer and At E13.0, the vestibule was isolated from the outside mesenchyme. Section sequences were reconstructed using only by a thin membrane (Fig. 5A and B). The tip of the TRI/3D-VOL software (Ratoc System Engineering, Tokyo, urorectal septum descended and extended into the vesti- Japan). bule. During the whole process from E11.5 to E13.0, the APOPTOSIS IN NORMAL MOUSE CLOACAL DEVELOPMENT 763

Fig. 1. Sagittal H&E sections (A–J) and schematic representation tubercle and cloacal regions were dramatically changed compared with (K–O) of the cloacal region of mouse embryos at E11.5 (A, F, K), E11.75 the other organs. Scale bars ϭ 1 mm (A–E) and 200 ␮m (F–J). Ac, anal (B, G, L), E12.0 (C, H, M), E12.5 (D, I, N), and E13.5 (E, J, O) around the canal; Cl, cloaca; Cm, cloacal membrane; Gt, genital tubercle; Hg, median plane. Low-magnification pictures show the whole embryo (A– hindgut; Pc, peritoneal cavity; Ta, tail; Tg, tailgut; Ur, urorectal septum; E), while high-magnification pictures show the corresponding cloacal Us, urogenital sinus.[Color figure can be viewed in the online issue, region (F–J). During these stages, shapes and relative sizes of the genital which is available at www.interscience.wiley.com]. urogenital sinus and distal end of the hindgut, i.e., the At E11.5 and E11.75, apoptotic cells and/or bodies were ventral and dorsal parts of the cloaca in the earlier stages, distributed in the epithelial layers and the underlying were constantly connected to each other. At E13.5, the mesenchyme region. In the underlying mesenchyme of the cloacal membrane that isolated the vestibule from the epithelial layer, the cells and/or bodies were particularly outside was completely disintegrated (Figs. 1E, J, and O abundant around the dorsal part of the cloaca, distal part and 5C). The vestibule was completely divided into the of the hindgut, and proximal part of the tailgut. At E11.5, ventral urogenital sulcus and the dorsal anorectal canal pyknotic cells in the epithelial layers were mainly distrib- by the extended tip of the urorectal septum. uted in the ventrocranial part of the cloacal membrane, dorsal part of the cloaca, distal part of the hindgut, and Spatiotemporal Distribution of Apoptosis proximal part of the tailgut. At E11.75, patterns of distri- During Cloacal Development bution were similar to those at E11.5, but at E11.75, cells In this study, H&E and TUNEL staining were used to were also clearly observed in the dorsal end of the cloacal analyze spatial and temporal distribution patterns of membrane. During these stages, cells were distributed in apoptosis during cloacal development. Distribution of the mesenchyme close to the epithelial layer containing intensely hematoxylin-stained granular substances was pyknotic cells, but distribution in mesenchyme was identical to that of structures labeled with TUNEL slightly less than in epithelial layers. staining in the adjacent section of the cloacal region At E12.0, pyknotic cells in epithelial layers were also (Fig. 2). Intensely hematoxylin-stained granules (py- distributed in the ventralmost part of the cloacal mem- knotic cells) were therefore almost confirmed as repre- brane. The distribution observed at E11.75 became con- senting apoptotic cells and/or bodies (Sasaki et al., centrated (Fig. 6B). In the median section at E12.0, py- 2001). The distribution of pyknotic cells in the cloacal knotic cells were clearly observed in the dorsalmost part of region from E11.5 to E13.0 was investigated. Three- the cloacal membrane and middle region in the epithelial dimensional reconstructions of serial H&E sections at layer between the cloacal membrane and entrance of the E11.5, E11.75, and E12.25 were made to determine the tailgut. In mesenchyme, the distribution was restricted to distributions of pyknotic cells (Fig. 6). the joint region between the hindgut and tailgut. At 764 SASAKI ET AL.

Fig. 2. Sagittal sections of the cloacal region at E11.5. In A, positional relationship among the hindgut, tailgut, and urorectal septum are shown by H&E staining. Most of the tailgut is not shown, but the point of entrance of the tailgut is present. In B and C, condensed chromatin could indicate apoptosis in sagittal sections of the cloacal region examined using H&E (B) and TUNEL staining (C). Apoptosis occurs in the epithelial layer (white arrowhead) of the urorectal septum and cloacal membrane and mesenchyme (black arrowhead) of the dorsal surface of ϭ ␮ the caudal hindgut. Scale bars 100 m. Fig. 4. Sagittal (A–D) and transverse (E) sections of cloacal region using H&E (A, C, and E) and TUNEL (B and D) staining at E12.5 (A and B) and E12.75 (C–E). Arrowheads indicate pyknotic cells (A and C) and apoptotic cells and/or bodies (B and D) in epithelium (white) and mesenchyme (black). E: Transverse section showing the canal between the urogenital sinus and distal hindgut. Section level is indicated by two asterisks in C. Scale bars ϭ 100 ␮m. Ta, tail.

Fig. 5. Sagittal section of cloacal region using H&E (A and C) and TUNEL (B) staining at E13.0 (A and B) and E13.5 (C). Arrowheads indicate pyknotic cells (A and C) and apoptotic cells and/or bodies (B) in epithelium (white) and mesenchyme (black). Scale bars ϭ 200 ␮m. Uc, urogenital sulcus; Ve, vestibule. Fig. 3. Sagittal sections of cloacal region stained with H&E at E12.0 (A and B) and E12.25 (C, D, and E). Arrowheads indicate pyknotic cells in epithelium (white) and mesenchyme (black). White arrows indicate the midpoint between the tailgut entrance and the cloacal membrane. At mesenchyme of the urorectal septum. According to the E12.0, pyknotic cells are not observed in the underlying mesenchyme of three-dimensional reconstruction from serial sagittal sec- the dorsal cloaca. At E12.25, pyknotic cells are observed in mesenchyme of the urorectal septum. At this point, epithelial cells starts to tions at E12.25, pyknotic cells in mesenchyme were situ- disintegrate. Scale bars ϭ 200 ␮m (A–D) and 50 ␮m (E). Cn, canal. ated in the urorectal septum mesenchyme ventral to the perineal cavity. From E12.5, the dorsalmost part of the cloacal membrane started to disintegrate under apoptosis (Fig. 4). E12.25, distribution in epithelial layers was similar to Apoptotic cells and/or bodies in the urorectal septum were that at E12.0. However, distribution in mesenchyme dif- located in a line, predominantly ventral to the peritoneal fered from those in embryos in earlier stages. In mesen- cavity. The extension of the line ran on the dorsalmost chyme of the ventral half of the urorectal septum, pyknotic part of the disintegrated cloacal membrane. In the epithe- cells displayed a linear distribution. Interestingly, py- lial layer of the hindgut, pyknotic cells were observed at knotic cells in the epithelial layer between the entrance of the midpoint between the tailgut entrance and cloacal the tailgut and the cloacal membrane and cells in the membrane at E12.0 and E12.25. During cloacal develop- dorsalmost part of the cloacal membrane were located ment, the distal end of the hindgut was continuously along a caudal extension of the line of pyknotic cells in the shifted ventrocaudally. By E12.5, the tailgut had disap- APOPTOSIS IN NORMAL MOUSE CLOACAL DEVELOPMENT 765 2000b, 2000c) mentioned that soon after fusion of the urorectal septum with the cloacal membrane, the urogenital and anal membranes begin to disintegrate. In con- trast, Politzer (1931) had already rejected the separation into anal and urogenital membranes. Many authors later reported that the urorectal septum does not actively de- scend in the direction of the cloacal membrane, and neither fusion of this septum with the membrane nor fusion of two lateral ridges of the cloacal wall occur (Van der Putte and Neeteson, 1983; Van der Putte, 1986; Kluth et al., 1995; Nievelstein et al., 1998; Paidas et al., 1999). The present study undertook minute examination of the nature of cloacal development using serial sagittal sections. Ventral and dorsal parts of the cloaca were divided by the superior urorectal septum. The superior urorectal septum and lateral ridges formed a canal at the border Fig. 6. Three-dimensional reconstruction of serial sagittal sections of between the ventral and dorsal parts. Interestingly, the cloacal region at E11.5 (A), E11.75 (B), and E12.25 (C). Blue lines indicate canal maintained a connection between the two parts un- lumen of the cloaca, urogenital sinus, hindgut, and tailgut. White lines til complete disappearance of the cloacal membrane at indicate outline of epithelial layers of the cloaca, urogenital sinus, hind- E13.5. During these processes, the cloacal membrane was gut, and tailgut. Red dots indicate apoptotic cells and/or bodies in not divided into urogenital and anal membranes, as Niev- mesenchymal regions. In addition, green dots indicate apoptotic cells elstein et al. (1998) reported. and/or bodies in epithelial layers. Many authors have mentioned that a shift of the dorsal cloaca or rectum is necessary to establish the anorectal canal (Bill and Johnson, 1958; Gans and Friedman, 1961; Van der Putte and Neeteson, 1983; Van der Putte, 1986). peared. According to the positional relationships among In the present study, from E11.5 to E12.5, ventral shift of the dorsalmost region of the cloacal membrane, the tip of the dorsal cloaca was observed. During these stages, the the urorectal septum, and midpoint between the rudimen- hindgut and tailgut migrate ventrally, and the urorectal tary tailgut entrance and cloacal membrane, the midpoint septum expanded ventrocaudally. These shifting pro- migrated ventrally to fuse with the cloacal membrane. cesses are considered to accompany development and Therefore, between this point and the tip of the urorectal growth of the genital tubercle. In addition, these processes septum, the cloacal membrane started to undergo apopto- also affect positional relationships between the entrance tic disintegration. of the tailgut, dorsal end of the cloacal membrane, and the At E13.0, the cloacal membrane was disintegrated by tip of the urorectal septum. Failure of these processes apoptosis to form the urogenital sulcus and anorectal ca- might thus cause anorectal malformation. nal (Fig. 5). Apoptotic cells and/or bodies were observed in Kluth et al. (1995) and Kluth and Lambrecht (1997) the mesenchyme of the urorectal septum ventral to the mentioned that in normal development, the area of the peritoneal cavity. At E13.5, pyknotic cells were aligned future anal orifice could be identified soon after establish- caudal to the peritoneal cavity. The line of pyknotic cells ment of the cloacal membrane in the dorsalmost region of in mesenchyme of the urorectal septum in those stages the membrane. In addition, the dorsal end of the cloacal was thus seen to migrate ventrocaudally according to membrane and the dorsal cloaca always remain in close growth of the urorectal septum and ventral migration of contact with the tail region, and this region carrying the the urogenital sulcus. primordial anal orifice is the fixed point in cloacal devel- DISCUSSION opment. In the early stages, the dorsal end of the cloacal membrane exists at the junction between the perineal Observation of Normal Cloacal Development region and tail bud. However, in later stages, the dorsal Formation of the urorectal septum and fusion with the end of the cloacal membrane is shifted ventrally, possibly cloacal membrane have been long debated in cloacal de- according to the development and growth of the external velopment. In order to explain the process of normal clo- genitalia. The point of the anal orifice might thus be acal developmental, the concept of the descending supe- altered by growth of the external genitalia. rior septum, or Tourneux fold (Tourneux, 1888), and two Spatiotemporal Distribution of Apoptosis in lateral ridges, or Rathke plicae (Rathke, 1832; Rettere, 1890), fusing to partition the cloaca was proposed long Cloacal Development ago. Many authors have supported the concept that one or Apoptosis is commonly observed during embryogenesis, two of these processes take place during cloacal develop- metamorphosis, or normal cell turnover, and apoptosis is ment (Keibel, 1895; Pohlman, 1911; De Vries and Fried- complementary to cell proliferation and differentiation in land, 1974a, 1974b; Stephens and Smith, 1988; Miller and morphogenesis and in the regulation of cell populations in Briglin, 1996; Kromer, 1999; Qi et al., 2000a, 2000b, embryos. 2000c). This means that the superior urorectal septum Cell death in developing systems has been clarified as reaches the cloacal membrane and divides the cloaca, and not merely a degenerative process, but rather an active the two lateral folds unite with the superior fold to form a and controlled phenomenon. In addition, for the formation complete septum. In addition, the cloacal membrane was of various structures during morphogenesis, cell death divided into separate urogenital and anal membranes by occurs according to precise temporal sequences and spa- the septum. Miller and Briglin (1996) and Qi et al. (2000a, tial patterns and is considered to play a key role by elim- 766 SASAKI ET AL. inating unnecessary cells to achieve complex histogenesis 2001). Target deletion of Shh, Gli2, or Hoxa13/Hoxd13 and organogenesis. For example, cell death is involved in results in the absence of external genitalia (Warot et al., remodeling the embryonic tail bud in humans (Kunimoto, 1997; Haraguchi et al., 2001; Perriton et al., 2002). On the 1918; Wittman et al., 1972; Fallon and Simandl, 1978), other hand, Fgf10 knockout mice show the absence of a mice (Wittman et al., 1972; Schoenwolf, 1984; Tam, 1984), glans (Haraguchi et al., 2000), while p63 null mice show rats (Butcher, 1929; Gajovic´ et al., 1989, 1993; Qi et al., abnormalities in the male and female urogenital tract and 2000b), and chicks (Klika and Jelinik, 1969; Van Horn, external genitalia (Yamada et al., 2003). In addition, Kim- 1971; Schoenwolf, 1981; Sanders et al., 1986; Mills and mel et al. (2000) reported Gli3 Ϫ/Ϫ mutants displayed Bellairs, 1989; Miller and Briglin, 1996). Several investi- anal stenosis and ectopic anus, while Gli2 Ϫ/Ϫ mutants gators have indicated that cell death is involved in re- showed imperforations and rectourethral fistula. moval of the tailgut in chicks (Van Horn, 1971), rats Numerous reports have suggested that apoptosis is as- (Sˇ vajger et al., 1985), and humans (Fallon and Simandl, sociated with down- or upregulation of various develop- 1978). Qi et al. (2000b) described the spatiotemporal dis- mental regulatory genes (Wyllie, 1987; Buttyan et al., tribution of apoptosis in cloacal development and reported 1988; Collins et al., 1994; Maas and Bei, 1997; Kera¨nen et the roles of apoptosis in tailgut regression, urorectal sep- al., 1999). Some evidence even suggests that all cells un- aration, urethral opening, and rupture of the anal mem- dergo apoptosis by default unless they are rescued by brane. In the present study, apoptosis in the epithelial survival factors (Raff, 1992; Steller, 1995). The develop- layers of the dorsal region resembled the findings of Qi et mental processes of the external genitalia might therefore al. (2000b). However, we noticed distribution of apoptotic be closely related to anorectal development, and the mo- cells and/or bodies (pyknotic cells) in mesenchyme. Distri- lecular mechanisms for development of the external gen- bution of pyknotic cells was plotted, and computer-as- italia might also control ventral shifting of the dorsal sisted three-dimensional reconstructions were created at cloaca and apoptosis in the cloacal region. Further de- E11.5, E11.75, and E12.25. These images elucidated spa- tailed studies of relationships between control mecha- tial and temporal distributions of pyknotic cells in mesen- nisms for the external genitalia and patterns of apoptosis chyme, although understanding is very difficult to achieve in the cloacal region would be informative for anorectal using only these sections. At E11.5 and E11.75, cells are development. mainly distributed around the dorsal part of the cloaca. However, at E12.25, in the mesenchyme around the dorsal LITERATURE CITED cloaca, cells were observed less frequently and were primarily distributed in the urorectal septum just ventral to Bill AH, Johnson RJ. 1958. Failure of migration of the rectal opening the peritoneal cavity. Interestingly, cells were arranged in as the cause for most cases of imperforate anus. Surg Gynecol Obstet 106:643–651. almost linear fashion in sagittal sections. In addition, the Butcher EO. 1929. The development of the somites in the white rat extension of the line ran to the region at which cells in the (Mus norvegicus albinus) and the fate of the myotomes, neural tube, epithelial layer disintegrated to form the vestibule, a fu- and gut in the tail. Am J Anat 44:381–439. ture cloacal opening. Those arrangements of pyknotic cells Buttyan R, Zakeri Z, Lickshin R, Wolgemuth D. 1988. 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