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Normal Development
In normal development at the beginning of the third week of gestation, the future
cloacal plate is discernible and composed of ectoderm and endoderm [16]. At the
beginning of the fourth week of gestation, the paired genital tubercle forms along
the upper part of the cloacal membrane. Fusion of the tubercles into a single genital
structure occurs at the fth week. Critical medial migration of mesenchymal cells
will give origin to the muscular and skeletal structures of the pelvis and abdominal
wall (Fig. 1.2).
Once the urorectal septum has grown sufciently in the caudal direction, cloacal
division into the bladder anlage and urogenital sinus ventrally and the rectum and
hindgut dorsally is complete by the seventh week. This normally occurs after the
aforementioned medial mesenchymal cell migration has been completed. The most
distal aspect of the septum then denes the origin of the urogenital and rectal openings (formerly the single cloacal opening), both of which are covered by the urogenital and anal membranes, respectively (formerly the single cloacal membrane)
(Fig.1.3).
Whether an extension of these migration processes also enables the later medial
closure or tubularization and ventralization of the urethra and spongiosal tissues and
their nal placement underneath the future corporal bodies is unknown. However, it
must be remembered that the even rarer occurrence of isolated penile epispadias
(without exstrophy) suggests that epispadias formation can be uncoupled from bladder exstrophy development, suggesting this may be governed by a separate mechanism (Figs.1.4 and 1.5).
Fig. 1.2 Early
embryological appearance
of the cloacal plate at
2weeks of gestation.
Sagittal image of embryo
Allantois
Mesenchymal body stalk
Yolk sac
Amnion
Neural plate
Primitive cloacal
plate
1 Epidemiology andEmbryology oftheBladder Exstrophy: Epispadias Complex
a
Neural tube
Cloacal region
Allantoic stalk
Allantoic umbilical
vessels in mesenchyme
of body stalk
off hind gut
Primitive streak
Cloacal plate
b
5
Urogenital sinus
Urorectal septum
Rectum
Fig. 1.3 (a) Cloacal plate begins the process of developing into the urorectal septum. (b) At
3 weeks of gestation, the septum will separate the urogenital sinus ventrally and the rectum
dorsally
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Urorectal septum
Allantois
N. Fernandez et al.
Genital tubercle
primordium
Postcloacal
gut
Cloacal membrane
Urogenital sinus
Umbilical artery
Dorsal aorta
Neural tube
Hind-gut
Rectal region of
cloaca
Fig. 1.4 Early formation of the genital tubercle and its relation to the cloacal membrane at
4weeks of gestation
Wall defect
Exstrophy
Genital tubercle
Bladder
Rectum
Aorta
Notochord
Anus
Neural tube
Fig. 1.5 Fiveweeks of gestation depicting the embryo with exstrophy and abdominal wall defect
1 Epidemiology andEmbryology oftheBladder Exstrophy: Epispadias Complex
7
It is important to point out that most of the embryologic etiologies for bladder
exstrophy remain largely conjecture or hypothesis. No animal studies of bladder
exstrophy to date have followed early embryonic development through to the late
embryo and fetus to conrm the typically described events leading to bladder
exstrophy. Nevertheless, some of the structural and molecular factors discussed
below have been found to be associated with bladder exstrophy in mammalian models and in patients with bladder exstrophy. Bladder exstrophy development has
essentially been centered around the caudal migration of the urorectal septum,
where most embryologic theories are based around physical descriptions of disordered medial migration of mesenchymal tissues destined to become muscle and
body wall and attempt to provide a rationale for this migration failure.
Historical Perspective andProposed Theories
The earliest events in bladder development are concerned with increasing cellular
differentiation into discrete mesenchymal (outer serosa, smooth muscle, and subepithelial lamina propria) and epithelial (uroepithelium) tissues. Additional cell
types also invest the bladder wall, ultimately comprising its vascular and neural
components, including resident cells such as macrophages with immunological or
other functions. Whether and how these components are rendered abnormal at the
cellular level during or after the earliest events leading to exstrophy and epispadias
are not completely understood. Some of the prevailing cellular theories are discussed later below.
The rst proposed hypothesis for bladder exstrophy was published by Patten and
Barry in 1952 [17]. They proposed that a low insertion of the genital tubercle in
relation to the cloacal membrane prevents normal mesodermal migration. This then
causes an abnormal or premature rupture of the cloacal membrane. One cited problem with this theory is that if the genital tubercle is forming more caudally, bladder
exstrophy should present regularly with penile structures in the perineal region,
which is not seen in most cases of bladder exstrophy-epispadias [16].
The most accepted embryologic theory proposes a “wedge effect” and was initially described in 1962 by Marshall and Muecke [18, 19]. In 1988, Mildenberger
elaborated on this idea, proposing that a caudal insertion of the embryo body stalk
prevents the interposition of the mesenchymal tissue in the midline [20]. In this
case, the most cephalad portion of the cloacal membrane remains in contact with the
lower portion of the body stalk, causing a wedge effect. This hypothesis is thought
to support the origin of severe OEIS cases.
Malrotation of pelvic ring primordia is another hypothesis proposed in 1997 by
Beaudoin etal. [21]. This theory involving skeletal maldevelopment was later supported by Kumar etal. hypothesizing that the well-known pubic diastasis of bladder
exstrophy is the inciting mechanism for bladder exstrophy formation as the principal disruptive phenomenon that occurs after organ formation [16]. They suggest that
the levator muscles, rather than encircling the bladder neck/anal sphincter complex,
form a hammock conguration due to the diastasis. The hammock becomes a
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N. Fernandez et al.
pushing force moving the hindgut anteriorly (hence anterior displacement of the
anus in exstrophy). The resulting progressive stretch on the remaining anterior bladder/developing penis structures leads to thinning (covered bladder exstrophy?) or
outright rupture (exposed bladder exstrophy) [16]. While this hypothesis remains to
be supported by any scientic evidence, it does add new consideration of the role of
muscular and skeletal structures in the etiology of bladder exstrophy.
Around the same time, Stec etal. published a hypothesis involving three steps or
opportunities for cellular dysfunction. Initially, premature rupture of the cloacal
membrane occurs, followed by a mechanical obstruction or failure in mesodermal
cell migration. This then results in cellular dysfunction leading to the anomaly [22],
although how these events are orchestrated is unknown.
Bladder Exstrophy Pathophysiology
The failure of medial mesenchymal cell migration between the ectoderm of the
abdomen and the cloaca is the most currently accepted pathophysiologic basis for
bladder exstrophy [4]. Marshall and Muecke reported that the center of the anomaly originates at the area where the future bladder neck should form [19, 21]. This
phenomenon occurs in the rst 4weeks of gestation, where an overdeveloped
cloacal membrane then physically prevents mesenchymal cell migration, resulting in premature cloacal membrane rupture [4]. Conversely, it is unclear if normal
mesenchymal migration is then preventing premature rupture of the cloacal membrane. Moreover, since the anus is often intact, normally developed, and patent,
though somewhat anteriorly displaced in simple bladder (non-cloacal) exstrophy,
this presumes the premature membrane rupture is limited to the urogenital membrane, sparing the anal membrane to open at the correct time. The timing of this
crucial step of membrane rupture is thought to generate the different subtypes of
the anomaly: isolated epispadias, bladder exstrophy, or—if the anal membrane or
early single cloacal membrane ruptures prematurely—OEIS. Nevertheless, it
remains paradoxical and unknown how a premature rupture of the anal membrane
still results in imperforate anus. Presumably, premature lesser ruptures of the cloacal membrane before the urorectal septum has completely reached the cloacal
membrane will result in an enterovesical stula. Regarding isolated epispadias
and membrane rupture, if only very distal/caudal membrane rupture occurs later
when mesenchymal migration has completed abdominal wall formation covering
the embryonic bladder but not yet contributed to tubularization/ventralization of
the urethra and spongiosum, this could underlie the formation of isolated
epispadias.
A rare subtype is the occult or so-called “covered” bladder exstrophy where a
musculoskeletal defect exists without visceral exstrophy. The exact prevalence of
this subtype has not been reported, and only isolated case reports have been published [23]. It is believed to be due to a sufcient mesodermal invasion into the
infraumbilical cloacal membrane to thinly close the body wall, but insufcient
deeper medial mesodermal migration to result in bladder closure itself [24].
1 Epidemiology andEmbryology oftheBladder Exstrophy: Epispadias Complex
9
Other theories of exstrophy development include an abnormal coordination of
spatial-temporal formation of the pelvic bones and bladder [4].
It bears repeating that the observed uncoupling of events such as body wall closure, bladder closure, and penile closure raises the possibility that distinct mechanisms are involved and/or that temporal or spatial processing of a more unied
process can be interrupted at various stages of development. Indeed, some authors
have challenged the concept of grouping epispadias, bladder exstrophy, and OEIS
together as a spectrum of a single condition. This is supported by the fact that the
original experiments were performed using chicken embryos, which possess a normal persistent cloaca postnatally and have no pubic symphysis [21]. Also, as mentioned earlier, there is no stage in embryonal development where cloacal membrane
rupture could physically result in displacement of the open urethral plate dorsally [21].
Newer Cellular Theories Underlying theBladder
Exstrophy-Epispadias Formation
While the above discussion has centered around anatomical and embryological
theories, it is even more important to consider candidates at the cell and molecular
level that may mediate the formation of bladder exstrophy-epispadias.
Sonic Hedgehog One of the key molecular steps in the normal development of the
bladder involves epithelial-mesenchymal interplay or interaction between the urothelium and the underlying undifferentiated mesenchyme, destined to form bladder
muscle (detrusor) [25, 26]. During normal bladder development, a group of signaling molecules becomes orchestrated by a protein known as sonic hedgehog (SHH).
The secretion of SHH by the urothelium activates a cascade or pathway of additional proteins (SHH→ Ptc1→ Gli2→ Bmp4) [27–29]. Acting together, this path-
way results in the mesenchyme differentiating into bladder smooth muscle cells.
Given the importance of these cellular events to bladder development, it is reasonable to hypothesize that spatial disruption of this pathway, particularly in the ventral aspect of the bladder, may be involved in the genesis of bladder exstrophy [27,
29, 30].
P63 A master regulator of epithelial development is the tumor suppressor protein
TP63, transcribed from the p63 gene and a member of the P53 tumor suppressor
family. The basal and intermediate layers of the urothelium express p63 during
organogenesis [31–33]. Since p63 is responsible for normal urothelial development
and function, and the urothelium is responsible for mesenchymal induction (see
above), this presents two opportunities for potential disruption of bladder development in exstrophy. Indeed, p63 mice engineered to lack p63 expression (p63
−/−
knockout mice) show apoptosis and decreased proliferation in the urothelium with
loss of urothelial smooth muscle development, particularly in the ventral bladder
wall [30, 34, 35] (Fig.1.6).
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N. Fernandez et al.
Fig. 1.6 (a) Histology of E18 wild-type mouse fetus. (b) p63
bladder wall. (Used with permission. Permission Pending)
−/−
mouse fetus with thin ventral
Indeed, a study comparing 163 patients (98% with classic bladder exstrophy)
and 285 ethnically matched controls revealed 7 single nucleotide polymorphisms
(SNPs) and 4 insertion/deletion (in/del) polymorphisms in a region of the p63 gene
promoter named ΔNP63. Importantly, ΔNP63 is a key regulator of anti-apoptosis in
the urothelium. While none of the SNPs were signicantly associated with bladder
exstrophy incidence, a statistically signicant increased risk of bladder exstrophyepispadias was associated with three out of four in/del ΔNP63 polymorphisms [36].
PERP and Desmosomes PERP (p53 apoptosis effector related to PMP-22) is a
plasma membrane protein. In humans, it is encoded by the PERP gene. PERP emerged
as a possible effector in bladder exstrophy from a gene expression proling study by Qi
etal. of human exstrophy and human and murine embryonic bladder tissues [37]. The
signicance of this nding is that the PERP promoter is bound not only by p53 but also
by p63 (see above), suggesting that PERP is regulated by p63 signaling [38, 39] P63PERP regulation indeed has a clinical correlate in at least one human syndrome [40].
Knockout studies reveal PERP is an essential component of the desmosome, a
cell complex critical for cells to adhere to each other in maintaining tissue integrity.
Without PERP, tissue integrity is compromised [41]. Furthermore, of the 162 differentially expressed genes identied in Qi’s gene proling study, 30% are associated with the desmosome. Three key components of the desmosome, desmoplakin
(DSP), desmin (DES) and desmulin (DMN) were the sixth most overexpressed
(DSP) and two most underexpressed (DES & DMN) genes, respectively, in the
bladder exstrophy samples [37]. Furthermore, multiple studies conrm that PERP
and desmosomal components work tightly together at the physical molecular level
to maintain the integrity of all epithelia [38, 41, 42].
Based on the above interactions drawn from human and knockout animal data,
Mahfuz etal. have proposed a unifying hypothesis that p63→ PERP↔ desmosomes may be a pathway of molecular epithelial and mesenchymal signaling disruption leading to bladder exstrophy formation [30].
1 Epidemiology andEmbryology oftheBladder Exstrophy: Epispadias Complex
11
Additional Molecular Candidates The WNT5A gene has been found upregulated 14-fold in bladder exstrophy patient samples. In murine cloacal mesoderm, mouse Gli2 overexpression induced the expression of Wnt5A, and in
cloacal ectoderm, Gli2 also up-regulated p63 expression. Together this allows
speculation that there is a common upstream pathway regulating both p63 and
Wnt5A [43].
A review by Hall etal. discusses additional candidate genes linked to bladder
exstrophy, including human chromosomal duplication in the region of 22q11.21
[44–46], which includes a novel heterozygous missense variant in the LZRT1 gene
[47]. A genome-wide association study on 110 bladder exstrophy patients vs. 1177
controls identied that changes in a locus involving a variant of ISL-1 (encoding the
insulin gene enhancer protein) on chromosome 5q11.1 were associated with
increased risk for bladder exstrophy [48].
Given the importance of the cloacal membrane discussed above, progenitor cells
adjacent to the cloacal mesenchyme are regulated by transcription factors Six1 and
Six2 and signaling by Dkk1. These factors are required for normal growth and
development of the perineum and could also be target molecular candidates in bladder exstrophy development [49, 50].
Kasprenski etal. (2020) reported reduced uroplakin-II (UPII) and p63 expression in specimens from the entire bladder exstrophy spectrum (CBE, CE, and epispadias) when compared to controls. Uroplakins are key molecules involved in
urothelial differentiation and permeability barrier development [51]. After surgical
closure, bladder exstrophy is also associated with a persistent increase in the expression of protein markers including CK13, CK29, UPIIIa, claudin 4, UPII, and p63
[52]. Whether or not these gene changes are solely related to bladder exposure to the
external environment or also regulate the genesis of the anomaly itself, they serve as
potential targets for developmental study.
Conclusion
The etiology of the bladder exstrophy and epispadias complex involves multiple
systems, including the urogenital, colorectal, and musculoskeletal tissues. Several
theories try to explain the physiopathology and embryology, but no consensus currently exists. The most critical embryological structures and events include the cloacal membrane, the timing of its rupture, the presence or absence of a urorectal
septum, and an abnormal migration of mesenchymal cells. Moreover, the molecular
aspects of this condition have also been studied with suspected involvement of the
SHH cascade and P63 master regulator inuence on mesenchymal induction.
Despite this considerable data, it is clear a great deal remains to be elucidated to
better understand the physiopathological, embryological, or molecular mechanisms
of bladder exstrophy development. Such insight may one day pave the way for
improved management of this challenging condition, or even its prevention
altogether.
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N. Fernandez et al.
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