Spatiotemporal distribution of apoptosis during normal cloacal development in mice.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 279A:761–767 (2004) Spatiotemporal Distribution of Apoptosis During Normal Cloacal Development in Mice 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 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 © 2004 WILEY-LISS, INC. 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 Grant sponsor: Grant-in-Aid for Scientiﬁc Research of the Ministry and Education, Culture, Sports, Science and Technology; Grant number: 13671639. *Correspondence to: Dr. Keiichi Akita, Unit of Clinical Anatomy, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo Tokyo 113-8519, Japan. Fax: 81-3-58030116. E-mail: email@example.com Received 5 February 2004; Accepted 4 May 2004 DOI 10.1002/ar.a.20062 Published online 7 July 2004 in Wiley InterScience (www.interscience.wiley.com). 762 SASAKI ET AL. 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 m 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. Three-Dimensional Reconstructions 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). RESULTS 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 APOPTOSIS IN NORMAL MOUSE CLOACAL DEVELOPMENT 763 Fig. 1. Sagittal H&E sections (A–J) and schematic representation (K–O) of the cloacal region of mouse embryos at E11.5 (A, F, K), E11.75 (B, G, L), E12.0 (C, H, M), E12.5 (D, I, N), and E13.5 (E, J, O) around the median plane. Low-magniﬁcation pictures show the whole embryo (A– E), while high-magniﬁcation pictures show the corresponding cloacal region (F–J). During these stages, shapes and relative sizes of the genital tubercle and cloacal regions were dramatically changed compared with the other organs. Scale bars ⫽ 1 mm (A–E) and 200 m (F–J). Ac, anal canal; Cl, cloaca; Cm, cloacal membrane; Gt, genital tubercle; Hg, hindgut; Pc, peritoneal cavity; Ta, tail; Tg, tailgut; Ur, urorectal septum; Us, urogenital sinus.[Color ﬁgure can be viewed in the online issue, which is available at www.interscience.wiley.com]. 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. 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 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. Threedimensional 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). 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 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 ⫽ 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 ⫽ 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 E12.0, pyknotic cells are not observed in the underlying mesenchyme of the dorsal cloaca. At E12.25, pyknotic cells are observed in mesenchyme of the urorectal septum. At this point, epithelial cells starts to disintegrate. Scale bars ⫽ 200 m (A–D) and 50 m (E). Cn, canal. 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 disap- APOPTOSIS IN NORMAL MOUSE CLOACAL DEVELOPMENT Fig. 6. Three-dimensional reconstruction of serial sagittal sections of cloacal region at E11.5 (A), E11.75 (B), and E12.25 (C). Blue lines indicate lumen of the cloaca, urogenital sinus, hindgut, and tailgut. White lines indicate outline of epithelial layers of the cloaca, urogenital sinus, hindgut, and tailgut. Red dots indicate apoptotic cells and/or bodies in mesenchymal regions. In addition, green dots indicate apoptotic cells and/or bodies in epithelial layers. peared. 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. DISCUSSION 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, 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 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 elim- 766 SASAKI ET AL. inating 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). 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