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Spatiotemporal distribution of apoptosis during normal cloacal development in mice.

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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: akita.fana@tmd.ac.jp
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).
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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).
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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-
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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). 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.
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