Spatiotemporal distribution of apoptosis during normal cloacal development in mice
Article first published online: 3 JUL 2004
Copyright © 2004 Wiley-Liss, Inc.
The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology
Volume 279A, Issue 2, pages 761–767, August 2004
How to Cite
Sasaki, C., Yamaguchi, K. and Akita, K. (2004), Spatiotemporal distribution of apoptosis during normal cloacal development in mice. Anat. Rec., 279A: 761–767. doi: 10.1002/ar.a.20062
- Issue published online: 19 JUL 2004
- Article first published online: 3 JUL 2004
- Manuscript Accepted: 4 MAY 2004
- Manuscript Received: 5 FEB 2004
- Grant-in-Aid for Scientific Research of the Ministry and Education, Culture, Sports, Science and Technology. Grant Number: 13671639
- normal cloacal development;
- mouse development;
- TUNEL method;
- genital tubercle
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.
Despite a long history of embryological research, the developmental processes of the anorectal canal remain contentious. The most debated point is whether fusion of the urorectal septum with the cloacal membrane occurs in normal development (Keibel, 1895; Pohlman, 1911; Politzer, 1931; De Vries and Friedland, 1974a, 1974b; Van der Putte and Neeteson, 1983; Van der Putte, 1986; Stephens and Smith, 1988; Kluth et al., 1995; Miller and Briglin, 1996; Nievelstein et al., 1998; Kromer, 1999; Paidas et al., 1999; Qi et al., 2000a, 2000b, 2000c). In addition, discussion has also centered around whether the 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 (E11.5) to E13.5 in detail, with particular focus on morphological changes in the dorsal part of the cloaca. According to the present findings, neither fusion of the urorectal septum with the cloacal membrane nor division of the cloacal membrane occurred, as mentioned by Nievelstein et al. (1998) in humans. Disintegration of the cloacal membrane is observed in one region, forming a vestibule into which the urogenital sinus and hindgut open. During cloacal developmental processes, ventral shift of the dorsal part of the cloaca and transformations of the distal part of the hindgut were also observed.
Such dramatic changes in the morphology and configuration of embryonic structures in the cloacal region are considered to be the result of embryonic cell differentiation, cell proliferation, and apoptosis (programmed cell death). Cell death is known to play an important role in the formation of various embryonic organs and is recognized as an important contributor to various areas integral to vertebrate development, such as limbs and digits (Saunders et al., 1962; Saunders and Fallon, 1967; Mori et al., 1995), heart (Pexieder, 1975), and tooth germs (Sasaki et al., 2001). The present study also examined distribution of apoptotic cells and bodies in the cloacal region. Distribution of apoptosis in the epithelial layers has already been reported by Qi et al. (2000b), and the present findings are similar. However, spatial and temporal distribution of apoptosis in the mesenchyme remains unclear. Computer-assisted three-dimensional reconstruction images were useful in understanding the spatial distributions of apoptosis, and dramatic changes in the distribution of apoptosis in mesenchyme were noted at approximately E12.0. Before this stage, distribution is primarily around the dorsal cloaca, but is subsequently found in the urorectal septum as a line that, when extended, passes through the area of disintegration of the cloacal membrane. This spatiotemporal distribution of apoptosis is deeply involved with the developmental processes of the cloaca, and possible roles of apoptosis in cloacal development are discussed herein.
MATERIALS AND METHODS
Animals and Tissue Preparation for Light Microscopy
Mature female ICR mice (SLC, Shizuoka, Japan) were mated overnight with a male mouse. The morning of the day on which a vaginal plug was found was designated as E0.5. For sequential examination of normal development in the cloacal region, two pregnant mice were sacrificed every 6 hr from E11.5 to E13.5 (n = 18). Handling of animals conformed to the guidelines for care and use of experimental animals as established by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (no. 10087). Embryos were fixed in 10% formalin, dehydrated, and embedded in paraffin. A total of 52 embryos were serially sectioned in the sagittal plane at 5 μm thickness. In two embryos at each stage, all sections were stained with hematoxylin and eosin (H&E). In all other embryos, sections were collected alternately, and one subset was stained with H&E.
In order to confirm the presence of apoptotic cells and bodies, nuclear DNA fragmentation of apoptotic cells in the other subset were labeled using TUNEL methods (Gavrieli et al., 1992) with an In Situ Cell Death Detection Kit (Roche Diagnostics, Tokyo, Japan). Peroxidase activity was visualized by immersion for 10 min in 0.02% diaminobenzidine (DAB) in 0.05 mol/l Tris-HCl buffer (pH 7.4) containing 0.01% H2O2. These sections were compared with adjacent H&E-stained sections to examine the distribution of apoptotic cells and bodies and intensely hematoxylin-stained granules.
Spatiotemporal distribution of pyknotic cells, apoptotic cells, and/or bodies was analyzed using computer-assisted three-dimensional reconstruction. Three-dimensional reconstructions were made from serial paraffin sections of E11.5, E11.75, and E12.0 specimens stained with H&E. All sections were photographed and epithelial layers were traced. Findings from sections with respect to the spatiotemporal distribution of granular substances intensely stained with H&E were marked, and distinction was made between granular substance in the epithelial layer and mesenchyme. Section sequences were reconstructed using TRI/3D-VOL software (Ratoc System Engineering, Tokyo, Japan).
Development and Growth of Genital Tubercle Affects Cloacal Development
We examined normal development of the cloacal region sequentially using serial sagittal sections of mouse embryos from E11.5 to E13.5 (Fig. 1A–J). In addition, schematic representation revealed development processes of the cloacal region based on the present studies. Cloacal development started before E11.5, and the cloaca was completely divided into the urogenital sinus and anorectal canal by E13.5. During these stages, the genital tubercle developed and grew substantially. Ventrocaudal outgrowth of the genital tubercle caused dorsocaudal rotation of the cloacal membrane. The cloaca shifted ventrocaudally according to genital tubercle development, and the distal part of the hindgut also shifted. In the cloaca, the urorectal septum was observed in sagittal sections, and the septum divided the cloaca into ventral and dorsal parts. During development, the ventral part of the cloaca was expanded ventrally according to development of the genital tubercle, whereas the dorsal part decreased in size to become the distal part of the hindgut, which could represent a precursor to the anorectal canal.
Ventral and Dorsal Parts of Cloaca Are Continuously Connected
As mentioned above, whether the urorectal septum fuses with the cloacal membrane has long been debated. We examined serial sagittal sections of mouse embryos during cloacal development to observe the border between the ventral and dorsal parts of the cloaca. At E11.5 and E11.75, no clear border between the two parts of the cloaca was apparent, and the distal part of the hindgut and proximal part of the tailgut opened into the dorsal wall of the cloaca (Figs. 1A, B, F, G, K, and L and 2). From E12.0 to E12.25, the urorectal septum was transformed according to ventral shifting of the hindgut and tailgut (Figs. 1C, H, and M and 3). The ventrocaudal part of the urorectal septum descended and expanded. The dorsal part of the cloaca became a distal part of the hindgut, and the tailgut began to disappear. Outgrowth of the septum made a small canal between the two parts of the cloaca by E12.25. The ceiling of the canal was formed by epithelial layers of the tip of the urorectal septum, and the floor of the canal was formed by the cloacal membrane.
At E12.5 and E12.75, the dorsalmost part of the cloacal membrane started to disintegrate (Figs. 1D, I, and N and 4). Disintegration occurred between the tip of the urorectal septum and the dorsal end of the cloacal membrane. The position of the dorsal end of the cloacal membrane was similar to that of the junction of the perineal region and tail bud until E12.25, but shifted ventrally thereafter. The canal was still observed between the urogenital sinus and distal part of the hindgut (Fig. 4E), but was so thin that only one section from each embryo showed this structure in the serial sagittal sections (Fig. 4B and D). Complete fusion of the septum and cloacal membrane was not observed. The cloacal membrane disintegrated in the distal end of the hindgut to form a small vestibule, and the canal and hindgut opened into this vestibule.
At E13.0, the vestibule was isolated from the outside only by a thin membrane (Fig. 5A and B). The tip of the urorectal septum descended and extended into the vestibule. During the whole process from E11.5 to E13.0, the urogenital sinus and distal end of the hindgut, i.e., the ventral and dorsal parts of the cloaca in the earlier stages, were constantly connected to each other. At E13.5, the cloacal membrane that isolated the vestibule from the outside was completely disintegrated (Figs. 1E, J, and O and 5C). The vestibule was completely divided into the ventral urogenital sulcus and the dorsal anorectal canal by the extended tip of the urorectal septum.
Spatiotemporal Distribution of Apoptosis During Cloacal Development
In this study, H&E and TUNEL staining were used to analyze spatial and temporal distribution patterns of apoptosis during cloacal development. Distribution of intensely hematoxylin-stained granular substances was identical to that of structures labeled with TUNEL staining in the adjacent section of the cloacal region (Fig. 2). Intensely hematoxylin-stained granules (pyknotic cells) were therefore almost confirmed as representing apoptotic cells and/or bodies (Sasaki et al., 2001). The distribution of pyknotic cells in the cloacal region from E11.5 to E13.0 was investigated. Three-dimensional reconstructions of serial H&E sections at E11.5, E11.75, and E12.25 were made to determine the distributions of pyknotic cells (Fig. 6).
At E11.5 and E11.75, apoptotic cells and/or bodies were distributed in the epithelial layers and the underlying mesenchyme region. In the underlying mesenchyme of the epithelial layer, the cells and/or bodies were particularly abundant around the dorsal part of the cloaca, distal part of the hindgut, and proximal part of the tailgut. At E11.5, pyknotic cells in the epithelial layers were mainly distributed in the ventrocranial part of the cloacal membrane, dorsal part of the cloaca, distal part of the hindgut, and proximal part of the tailgut. At E11.75, patterns of distribution were similar to those at E11.5, but at E11.75, cells were also clearly observed in the dorsal end of the cloacal membrane. During these stages, cells were distributed in the mesenchyme close to the epithelial layer containing pyknotic cells, but distribution in mesenchyme was slightly less than in epithelial layers.
At E12.0, pyknotic cells in epithelial layers were also distributed in the ventralmost part of the cloacal membrane. The distribution observed at E11.75 became concentrated (Fig. 6B). In the median section at E12.0, pyknotic cells were clearly observed in the dorsalmost part of the cloacal membrane and middle region in the epithelial layer between the cloacal membrane and entrance of the tailgut. In mesenchyme, the distribution was restricted to the joint region between the hindgut and tailgut. At E12.25, distribution in epithelial layers was similar to that at E12.0. However, distribution in mesenchyme differed from those in embryos in earlier stages. In mesenchyme of the ventral half of the urorectal septum, pyknotic cells displayed a linear distribution. Interestingly, pyknotic cells in the epithelial layer between the entrance of the tailgut and the cloacal membrane and cells in the dorsalmost part of the cloacal membrane were located along a caudal extension of the line of pyknotic cells in the mesenchyme of the urorectal septum. According to the three-dimensional reconstruction from serial sagittal sections at E12.25, pyknotic cells in mesenchyme were situated 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). Apoptotic cells and/or bodies in the urorectal septum were located in a line, predominantly ventral to the peritoneal cavity. The extension of the line ran on the dorsalmost part of the disintegrated cloacal membrane. In the epithelial layer of the hindgut, pyknotic cells were observed at the midpoint between the tailgut entrance and cloacal membrane at E12.0 and E12.25. During cloacal development, the distal end of the hindgut was continuously shifted ventrocaudally. By E12.5, the tailgut had disappeared. According to the positional relationships among the dorsalmost region of the cloacal membrane, the tip of the urorectal septum, and midpoint between the rudimentary tailgut entrance and cloacal membrane, the midpoint migrated ventrally to fuse with the cloacal membrane. Therefore, between this point and the tip of the urorectal septum, the cloacal membrane started to undergo apoptotic disintegration.
At E13.0, the cloacal membrane was disintegrated by apoptosis to form the urogenital sulcus and anorectal canal (Fig. 5). Apoptotic cells and/or bodies were observed in the mesenchyme of the urorectal septum ventral to the peritoneal cavity. At E13.5, pyknotic cells were aligned caudal to the peritoneal cavity. The line of pyknotic cells in mesenchyme of the urorectal septum in those stages was thus seen to migrate ventrocaudally according to growth of the urorectal septum and ventral migration of the urogenital sulcus.
Observation of Normal Cloacal Development
Formation of the urorectal septum and fusion with the cloacal membrane have been long debated in cloacal development. In order to explain the process of normal cloacal developmental, the concept of the descending superior septum, or Tourneux fold (Tourneux, 1888), and two lateral ridges, or Rathke plicae (Rathke, 1832; Rettere, 1890), fusing to partition the cloaca was proposed long ago. Many authors have supported the concept that one or two of these processes take place during cloacal development (Keibel, 1895; Pohlman, 1911; De Vries and Friedland, 1974a, 1974b; Stephens and Smith, 1988; Miller and Briglin, 1996; Kromer, 1999; Qi et al., 2000a, 2000b, 2000c). This means that the superior urorectal septum reaches the cloacal membrane and divides the cloaca, and the two lateral folds unite with the superior fold to form a complete septum. In addition, the cloacal membrane was divided into separate urogenital and anal membranes by the septum. Miller and Briglin (1996) and Qi et al. (2000a, 2000b, 2000c) mentioned that soon after fusion of the urorectal septum with the cloacal membrane, the urogenital and anal membranes begin to disintegrate. In contrast, Politzer (1931) had already rejected the separation into anal and urogenital membranes. Many authors later reported that the urorectal septum does not actively descend 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 between the ventral and dorsal parts. Interestingly, the canal maintained a connection between the two parts until complete disappearance of the cloacal membrane at E13.5. During these processes, the cloacal membrane was not divided into urogenital and anal membranes, as Nievelstein et al. (1998) reported.
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). In the present study, from E11.5 to E12.5, ventral shift of the dorsal cloaca was observed. During these stages, the hindgut and tailgut migrate ventrally, and the urorectal septum expanded ventrocaudally. These shifting processes are considered to accompany development and growth of the genital tubercle. In addition, these processes also affect positional relationships between the entrance of the tailgut, dorsal end of the cloacal membrane, and the tip of the urorectal septum. Failure of these processes might thus cause anorectal malformation.
Kluth et al. (1995) and Kluth and Lambrecht (1997) mentioned that in normal development, the area of the future anal orifice could be identified soon after establishment of the cloacal membrane in the dorsalmost region of the membrane. In addition, the dorsal end of the cloacal membrane and the dorsal cloaca always remain in close contact with the tail region, and this region carrying the primordial anal orifice is the fixed point in cloacal development. In the early stages, the dorsal end of the cloacal membrane exists at the junction between the perineal region and tail bud. However, in later stages, the dorsal end of the cloacal membrane is shifted ventrally, possibly according to the development and growth of the external genitalia. The point of the anal orifice might thus be altered by growth of the external genitalia.
Spatiotemporal Distribution of Apoptosis in Cloacal Development
Apoptosis is commonly observed during embryogenesis, metamorphosis, or normal cell turnover, and apoptosis is complementary to cell proliferation and differentiation in morphogenesis and in the regulation of cell populations in embryos.
Cell death in developing systems has been clarified as not merely a degenerative process, but rather an active and controlled phenomenon. In addition, for the formation of various structures during morphogenesis, cell death occurs according to precise temporal sequences and spatial patterns and is considered to play a key role by eliminating unnecessary cells to achieve complex histogenesis and organogenesis. For example, cell death is involved in remodeling the embryonic tail bud in humans (Kunimoto, 1918; Wittman et al., 1972; Fallon and Simandl, 1978), mice (Wittman et al., 1972; Schoenwolf, 1984; Tam, 1984), rats (Butcher, 1929; Gajović et al., 1989, 1993; Qi et al., 2000b), and chicks (Klika and Jelinik, 1969; Van Horn, 1971; Schoenwolf, 1981; Sanders et al., 1986; Mills and Bellairs, 1989; Miller and Briglin, 1996). Several investigators have indicated that cell death is involved in removal of the tailgut in chicks (Van Horn, 1971), rats (Švajger et al., 1985), and humans (Fallon and Simandl, 1978). Qi et al. (2000b) described the spatiotemporal distribution of apoptosis in cloacal development and reported the roles of apoptosis in tailgut regression, urorectal separation, urethral opening, and rupture of the anal membrane. In the present study, apoptosis in the epithelial layers of the dorsal region resembled the findings of Qi et al. (2000b). However, we noticed distribution of apoptotic cells and/or bodies (pyknotic cells) in mesenchyme. Distribution of pyknotic cells was plotted, and computer-assisted three-dimensional reconstructions were created at E11.5, E11.75, and E12.25. These images elucidated spatial and temporal distributions of pyknotic cells in mesenchyme, although understanding is very difficult to achieve using only these sections. At E11.5 and E11.75, cells are mainly distributed around the dorsal part of the cloaca. However, at E12.25, in the mesenchyme around the dorsal cloaca, cells were observed less frequently and were primarily distributed in the urorectal septum just ventral to the peritoneal cavity. Interestingly, cells were arranged in almost linear fashion in sagittal sections. In addition, the extension of the line ran to the region at which cells in the epithelial layer disintegrated to form the vestibule, a future cloacal opening. Those arrangements of pyknotic cells were observed until E13.5. The stages of cloacal development might thus be classified into two phases according to the distribution of pyknotic cells in mesenchyme. The critical stage might be E12.0 (Fig. 3A), when cells were barely observed in mesenchyme of the urorectal septum and the region around the dorsal cloaca.
During cloacal development, from E11.5 to E12.5, the ventral shift of the hindgut is observed. The patterns of apoptosis in mesenchyme around the dorsal part of the cloaca might occur ahead of the actual transformation in this part. The abundant pyknotic cells might therefore be associated with transformation of the dorsal cloaca. Conversely, the line of pyknotic cells from E12.25 might be related to transformation of the urorectal septum and disintegration of the cloacal membrane. However, the reason for apoptosis occurring only in the ventral part of the septum is unknown. Future studies should examine relationships between developmental control mechanisms of the urorectal septum and formation of the cloacal opening. In addition, further studies will attempt to identify signaling molecules in this area to understand formation of the anorectal canal.
The molecular mechanisms behind development of the mammalian external genitalia have recently been reported. During development of the genital tubercle, surface ectoderm cells expressing both Fgf8 and Shh regulate the outgrowth. Fgf8 controls the expression of Fgf10, Hoxd13, Msx1, and Bmp4 in the underlying mesenchyme (Haraguchi et al., 2000). Conversely, Shh can regulate Ptch1, Bmp4, Hoxd13, Gli1, and Fgf10 (Haraguchi et al., 2001). Target deletion of Shh, Gli2, or Hoxa13/Hoxd13 results in the absence of external genitalia (Warot et al., 1997; Haraguchi et al., 2001; Perriton et al., 2002). On the other hand, Fgf10 knockout mice show the absence of a glans (Haraguchi et al., 2000), while p63 null mice show abnormalities in the male and female urogenital tract and external genitalia (Yamada et al., 2003). In addition, Kimmel et al. (2000) reported Gli3 −/− mutants displayed anal stenosis and ectopic anus, while Gli2 −/− mutants showed imperforations and rectourethral fistula.
Numerous reports have suggested that apoptosis is associated with down- or upregulation of various developmental regulatory genes (Wyllie, 1987; Buttyan et al., 1988; Collins et al., 1994; Maas and Bei, 1997; Keränen et al., 1999). Some evidence even suggests that all cells undergo apoptosis by default unless they are rescued by survival factors (Raff, 1992; Steller, 1995). The developmental processes of the external genitalia might therefore be closely related to anorectal development, and the molecular mechanisms for development of the external genitalia might also control ventral shifting of the dorsal cloaca and apoptosis in the cloacal region. Further detailed studies of relationships between control mechanisms for the external genitalia and patterns of apoptosis in the cloacal region would be informative for anorectal development.
- 1958. Failure of migration of the rectal opening as the cause for most cases of imperforate anus. Surg Gynecol Obstet 106: 643–651. , .
- 1929. The development of the somites in the white rat (Mus norvegicus albinus) and the fate of the myotomes, neural tube, and gut in the tail. Am J Anat 44: 381–439. .
- 1988. Cascade induction of c-fos, c-myc, and heat shock 70 K transcripts during regression of the rat ventral prostate gland. Mol Endocrinol 2: 650–657. , , , .
- 1994. Growth factors as survival factors: regulation of apoptosis. BioEssays 16: 133–138. , , , , .
- 1974a. The staged development of the anus and rectum in human embryos and fetuses. J Pediatr Surg 9: 755–769. , .
- 1974b. Congenital “H-type” ano-urethral fistula. Radiology 113: 387–407. , .
- 1978. Evidence of a role for cell death in the disappearance of the embryonic human tail. Am J Anat 152: 111–130. , .
- 1989. Origin of the notochord in the rat embryo tail. Anat Embryol 179: 305–310. , , .
- 1993. Morphological evidence for secondary formation of the tail gut in the rat embryo. Anat Embryol 187: 291–297. , , .
- 1961. Some new concepts in the embryology, anatomy, physiology and surgical correction of imperforate anus. West J Surg Obstet Gynecol 63: 34–37. , .
- 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119: 493–501. , , .
- 2000. Molecular analysis of external genitalia formation: the role of fibroblast growth factor (Fgf) genes during genital tubercle formation. Development 127: 2471–2479. , , , , , , , , , , .
- 2001. Unique functions of Sonic hedgehog signaling during external genitalia development. Development 128: 4241–4250. , , , , , , , .
- 1895. Ueber die Entwickelung von Harnblase, Harnröhre und Damm beim Menschen. Verh Anat Gesellsch. p 189–199. .
- 1999. Gene expression patterns associated with suppression of odontogenesis in mouse and vole diastema region. Dev Genes Evol 209: 495–506. , , , , .
- 2000. New mouse models of congenital anorectal malformations. J Pediatr Surg 35: 227–230. , , , .
- 1969. The structure of the end and tail bud of the chick embryo. Folia Morphol 17: 29–40. , .
- 1995. The principle of normal and abnormal hindgut development. J Pediatr Surg 30: 1143–1147. , , .
- 1997. Current concepts in the embryology of anorectal malformations. Semin Pediatr Surg 6: 180–186. , .
- 1999. Further study of the urorectal septum in staged human embryos. Folia Morphol (Warsz) 58: 53. .
- 1918. The development and reduction of the tail and of the caudal end of the spinal cord. Carnegie Contr Embryol 8: 161–203. .
- 1997. The genetic control of early tooth development. Crit Rev Oral Biol Med 8: 4–39. , .
- 1996. Apoptosis removes chick tail gut and remnant of the primitive streak. Dev Dynamic 206: 212–218. , .
- 1989. Mitosis and cell death in the tail of the chick embryo. Anat Embryol 180: 301–308. , .
- 1995. Programmed cell death in the interdigital tissue of the fetal mouse limb is apoptosis with DNA fragmentation. Anat Rec 242: 103–110. , , , , , .
- 1998. Normal and abnormal embryonic development of the anorectum in human embryos. Teratology 57: 70–78. , , , , .
- 1999. Septation and differentiation of the embryonic human cloaca. J Pediatr Surg 34: 877–884. , , , , .
- 2002. Sonic hedgehog signaling from the urethral epithelium controls external genital development. Development 247: 26–46. , , , , .
- 1975. Cell death in the morphogenesis and teratogenesis of the heart. Adv Anat Embryol Cell Biol 51: 7–99. .
- 1911. The development of the cloaca in human embryos. Am J Anat 12: 1–26. .
- 1931. Über die Entwicklung des Dammes beim Menschen. Z Ges Anat I Abt 95: 734–768. .
- 2000a. Does the urorectal septum fuse with the cloacal membrane? J Urol 164: 2070–2072. , , , .
- 2000b. Clarification of the process of separation of the cloaca into rectum and urogenital sinus in the rat embryo. J Pediatr Surg 35: 1810–1816. , , , .
- 2000c. Apoptosis during regression of the tailgut and septation of the cloaca. J Pediatric Surg 35: 1556–1561. , , , .
- 1992. Social controls on cell survival and cell death. Nature 356: 397–400. .
- 1832. Abhandlungen zur Bildungs und Entwicklungsgeschichte der Tiere. Leipzig: F.C.W. Vogel. .
- 1890. Sur l'origine et l'évolution de la région ano-génitale des mammifères. J Ana Physiol 26: 126–216. .
- 1986. An experimental and morphological analysis of the tail bud mesenchyme of the chick embryo. Anat Embryol 174: 179–185. , , , .
- 2001. Apoptosis in regressive deciduous tooth germs of Suncus murinus evaluated by the TUNEL method and electron microscopy. Arch Oral Biol 46: 649–660. , , .
- 1962. Cellular death in morphogenesis of the avian wing. Dev Biol 5: 147–178. , , .
- 1967. Cell death in morphogenesis. In: LockeM, editor. Major problems in developmental biology. New York: Academic Press. p 289–314. , .
- 1981. Morphogenetic processes involved in the remodeling of the tail region of the chick embryo. Anat Embryol 162: 183–197. .
- 1984. Histological and ultrastructural studies of secondary neurulation in mouse embryos. Am J Anat 169: 361–376. .
- 1995. Mechanisms and genes of cellular suicide. Science 267: 1445–1449. .
- 1988. Anorectal malformations in children. Birth Defects Orig Artic Ser 24: 177. , .
- 1985. Tail gut formation in the rat embryo. Roux's Arch Dev Biol 194: 429–432. , , , .
- 1984. The histogenetic capacity of tissues in the caudal end of the embryonic axis of the mouse. J Embryol Exp Morphol 82: 253–266. .
- 1888. Sur les premieres développements du cloaques du tubercule génital et de l'anus chez l'embyon de mouton. J Anat 24: 503–517. .
- 1983. The normal development of the anorectum in the pig. Acta Morphol Neerl Scand 21: 107–132. , .
- 1986. Normal and abnormal development of the anorectum. J Pediatr Surg 21: 434–440. .
- 1971. A study of the degeneration of the tailgut in the chick embryo. Acta Morphol Neerl Scand 8: 234–235. .
- 1997. Gene dosage-dependent effects of the Hoxa-13 and Hoxd-13 mutations on morphogenesis of the terminal parts of the digestive and urogenital tracts. Development 124: 4781–4791. , , , , .
- 1972. Electron microscopy and histochemistry of tail regression in the Brachyury mouse. Dev Biol 27: 419–424. , , , .
- 1987. Apoptosis: cell death in tissue regulation. J Pathol 153: 313–316. .
- 2003. Cellular and molecular mechanisms of development of the external genitalia. Differentiation 71: 445–460. , , , .