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.