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Animal Models |
From the Program in Developmental Biology,*
Research
Institute, The Hospital for Sick Children, and Department of Molecular
and Medical Genetics, University of Toronto, Toronto, Ontario, Canada;
the Program in Infection, Immunity, Injury, and
Repair,
Research Institute, The Hospital for
Sick Children, and Department of Surgery, University of Toronto,
Toronto, Ontario, Canada; and the Department of Cell
Biology,
Vanderbilt University Medical
Center, Nashville, Tennessee
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several congenital syndromes have been associated with malformations affecting the development of anus and rectum, including Currarino syndrome and Townes-Brocks syndrome.5-8 The Currarino syndrome (MIM no. 176450) is an autosomal-dominant genetic disorder characterized by the partial agenesis of sacral vertebrae, presacral teratoma, and anorectal malformations. Linkage and mutational analyses have identified the major locus for Currarino syndrome to be a homeobox gene, HLXB9, located on chromosome 7q36.5-7 However, the exact role of HLXB9 in anorectal malformations is unclear because Hlxb9 mutant mice do not display any anorectal, genitourinary, or sacral disturbances.9,10 Townes-Brocks syndrome (MIM no. 107480) is a rare dominant malformation syndrome, with a combination of anal, renal, limb, and ear anomalies, caused by mutations in the SALL1 zinc-finger protein.8 The VACTERL syndrome, which represents a nonrandom association of vertebral, anorectal, cardiac, tracheossophageal, renal, and limb anomalies includes another important cohort of patients with anorectal malformations.11,12 Among these anomalies, malformations involving foregut and hindgut development are highly associated.11,12 The genetic basis of the VACTERL syndrome is currently unknown.
Development of anus and rectum from the distal (or posterior) hindgut has been described in normal human fetuses at different stages.13 Similar studies on anorectal malformations in human fetuses, however, have not been performed because of the lack of available tissue. Several murine models are known to display various anorectal malformations but, most importantly, the naturally occurring Sd mouse (Danforth’s short-tail mouse).14-17 These mice have an autosomal-dominant trait for imperforate anus and pass on this semi-dominant trait with high penetrance, where all of the homozygotes and most of the heterozygotes are affected. To date, the gene defective in Sd mice has yet to be identified. Domestic pigs have a naturally high incidence of anorectal malformation and can be bred to generate an animal model to study the anatomy and embryology of anorectal malformation.18-20 Like the Sd mice, however, the specific genetic mechanism for the anorectal malformation is not known. In both mice and rats, several teratogenic models were discovered in which anorectal malformation occur frequently using agents such as etretinate, retinoic acid, or adriamycin.21-23 They have accompanying abnormalities involving the skeletal and genitourinary systems. The specific gene(s) influenced by these teratogens is not known.
Recently, mutant mice involving Sonic hedgehog (Shh), a secreted signaling molecule that plays diverse roles in vertebrate development,24,25 were shown to display an imperforate anus phenotype,26 in addition to other anomalies including complete agenesis of the vertebral column, heart-looping defects, tracheoesophageal fistula, and loss of distal limb structures.27-33 Shh is expressed throughout the rostral-caudal extent of the gut endoderm and has been implicated in the first phase of signaling from the endoderm to the mesoderm.34-37 A role for Shh signaling in foregut development has been reported.32,33,38,39 Shh null mice exhibit esophageal atresia/stenosis, tracheoesophageal fistula, and lung anomalies indicating that Shh signaling is required for the normal development of esophagus, trachea, and lung.32,33 Perturbation of Shh signaling in transgenic mice and in chick embryos also affects the development of the pancreas, another derivative of the foregut.38,39 However, the exact role of Shh in hindgut development is not known. Shh is specifically expressed in the endoderm of the developing hindgut and embryological studies in chick embryos reveal that overexpression of Shh can induce the expression of Bmp4 and Hoxd13 in hindgut mesoderm.34 The initial observations of hindgut abnormalities in Shh null mice and in mice defective in Shh transcription factors, Gli2 and Gli3 prompted us to investigate the role of Shh signaling pathway in anorectal malformations in these mutant mice.
In this study, we performed a systematic analysis of the distal hindgut phenotypes in Shh null mice as well as in mutant mice that are deficient for Gli2 and/or Gli3.29,40-42 We report here the strongest genetic association thus far for a developmental pathway and anorectal malformations in mice. These mutant mice display a spectrum of anorectal malformations similar to that observed in humans. These observations establish the role of Shh signaling in the development of the distal hindgut and suggest that mutations affecting Shh signaling might be involved in anorectal malformations in humans.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gli2 mutant mice carry a targeted deletion of the DNA-binding zinc-finger motifs of the gene.24 Gli3 mutant mice are spontaneous null mutants with a large 3' deletion of the gene.25 Gli2+/-, Gli3+/-, and Gli2+/-;Gli3+/- mice were maintained in a mixed 129 and CD1 background. Intercrosses of Gli2+/-;Gli3+/- mice were used to generate Gli2-/-;Gli3+/-, Gli2+/-;Gli3-/- and Gli2-/-;Gli3-/- embryos. Shh mutant mice carry a targeted deletion of most of the protein-coding region of the gene.29 The generation of mutant embryos were performed as described previously.29,41,43 Genotype of mutant embryos was determined by polymerase chain reaction analysis of yolk sac DNA.
Dissection, Histology, and Paraffin Embedding
Midday of the day of the vaginal plug was considered as E0.5 in the timing of the embryo collection. Embryos were dissected out and fixed in 4% paraformaldehyde overnight at 4°C. They were dehydrated, processed, and embedded in paraffin wax before sectioning at 7 µm. Slides were then dewaxed, rehydrated, and stained with hematoxylin and eosin.
Microdissection and Radiography
Embryos were aseptically dissected from uterine decidua and staged by external features at E14.5 and 18.5. Omnipaque radiographic dye was injected into the distal intestine of each embryo and standard anteroposterior radiographs were taken.
Scanning Electron Microscopy (SEM)
Dissected embryos were fixed in osmium tetroxide, dehydrated in ethanol, mounted with carbon paint, and examined on JEOL820 SEM as described.44
Histology and in Situ Hybridization
Whole-mount in situ hybridization was performed according to the standard protocol.43,45,46 Section in situ was performed according to published procedures.41,47 The probes used were Gli1, Gli2, and Gli3.48
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Results |
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In mice, the first external sign of developing anus,
rectum, and lower urinary tract is the appearance of urethral and anal
orifices in the tail groove of the perineum between the hindlimb buds
and tail on E14.5 (boxed area in Figure 1; a–d
). Between E12.5 and E13.5, the
most distal hindgut and a more anteriorly located urethral compartment
at the base of genital swelling, share a residual common cloacal cavity
(marked as c in Figure 1e
). The cavity is covered by a thin cloacal
membrane, which degenerates at approximately E14.5 (Figure 1g)
. By SEM,
the anal orifice can be easily identified by the cobbled appearance of
the villi in the anorectal mucosa and the urethral orifices by the
ridged surface of the uroepithelium (Figure 1d
; data not shown). The
onset of the partition of the cloaca into the ventral urogenital sinus
and dorsal anorectum is observed clearly by E10.5 (Figure 1e)
. However,
a complete partition between the lower urinary and intestinal tracts
does not occur until E14.5 when anus and urethra open independently
into the perineum (Figure 1g)
. The appearance of male and female
external genitalia in the perineum remains indistinguishable on E14.5.
Between E14.5 and birth (E19.5 to E20.5), the distance between the anal
and urethral orifices lengthens with the growth of genital tubercle and
swellings (Figure 1, g and h)
revealing more characteristic male
(Figure 2a)
and female (Figure 3a)
external features at birth. The three
Shh-responsive Gli transcription factors (Gli1, Gli2, and Gli3) are all
highly expressed in the visceral mesoderm of the developing hindgut
suggesting that Shh signaling might play a critical role in hindgut
development (Figure 1
; i–l).
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To determine the role of Shh in hindgut development, we analyzed
E14.5 (n = 5) and E18.5
(n = 7) Shh-/- mutants
by SEM, light microscopy, and histological examination. Strikingly, all
Shh-/- mutants displayed a persistent cloaca
where the lower urinary tract and anorectum share a common outlet.
E18.5 Shh-/- mutants exhibited a single poorly
developed perineal opening (Figure 2b)
similar to those observed in
human cloacal malformations in which the perineal area lacks any
obvious external features of genitalia and is partially covered by
amniotic membrane (see Figure 2c
for example). The dissected autopsy
specimen from the same patient clearly demonstrates a persistent cloaca
with distal intestine and ureters draining into a common cavity (Figure 2f)
. Histological analysis of E14.5 Shh-/-
mutants reveals a similar condition with a ventrally located
hypoplastic bladder and an intestinal tract draining into a common
cloacal channel, which is joined by shortened ureters (Figure 2e)
. SEM
and histological examination revealed that the mucosal architecture of
the distal intestine is colonic suggesting epithelial colonic
differentiation occurs in the proximal hindgut of
Shh-/- mutants (data not shown). These
observations indicate that Shh signaling is essential for the
development of the distal hindgut, including the rectum and anus.
Imperforate Anus in Gli2 Mutant Mice
We examined E14.5 (n = 7) and E18.5
(n = 30) Gli2-/- mice to
determine the function of Gli2 in hindgut development. All
Gli2-/- mice displayed an imperforate anus
(absence of anus and lower rectum) with recto-urethral/recto-vaginal
fistula and exhibited a single urethral opening in the perineum (Figure 3, b and e)
.42
The fistula is found between the distal
intestine and bladder (Figure 3e)
and can be clearly shown by the
injection of a radiographic contrast dye into the distal intestine
(Figure 3h)
. In wild-type mice, the radiographic contrast dye outlines
only the rectum and anus (Figure 3g)
. These data demonstrate that
mice lacking Gli2 exhibit a severe anorectal defect that is milder than
the cloacal phenotype of Shh null mice. The distal hindgut anomalies
found in Gli2-/- mice are identical to the
anorectal agenesis with recto-prostatic urethral/recto-vaginal fistula
commonly seen in humans.
Anal Stenosis in Gli3 Mutant Mice
Gli3-/- mice have a subtle distal hindgut
phenotype (n = 29). Although their bladder,
urethra, and rectum developed normally, the anus in all
Gli3-/- mice examined is significantly narrower
by 30% as compared to that of wild-type mice (Figure 3; c, f, and i
).
In addition, in approximately one third of the
Gli3-/- mice, the anus appears ectopic with a
location more ventral at the junction between the lower abdomen and
tail (data not shown).
Embryology of Anorectal Malformation
The onset of normal development of ventral urogenital and dorsal
anorectum from the proximal cloaca is first observed between E11.5 and
E12.5. To determine whether anorectal malformation observed in
Shh and Gli2 mutants are because of failure in
the development of a septum, which partitions the cloaca into ventral
urogenital sinus and dorsal anorectum, we specifically examined the
sagittal and transverse sections of E11.5 and E12.5 embryos from
wild-type, Gli2, and Shh mutant embryos. These
analyses reveal that the partitioning of cloaca into ventral urogenital
and dorsal anorectal tracts does not occur by development of a septum
as previously suggested.49,50
Presumptive urorectal septum
on sagittal images is not corroborated by any anatomical equivalent on
the transverse sections in wild-type embryos (n
= 4) (Figure 4; a, b, e, and f
).
Furthermore, on transverse sections, there was no evidence of lateral
ridge formation or fusion during normal development of anus and rectum
in wild-type embryos (n = 4) (Figure 4b)
. These
observations suggests that the development of dorsal anorectum and
ventral urogenital tract from the cloaca is likely because of an
anterior-to-posterior progression of regional specification and
differentiation of these structures by endodermal-mesenchymal
interaction.51
In the absence of either the Shh signaling
or Gli2, the major transducer of Shh signaling, the most posterior end
of the hindgut fails to differentiate into anorectum whereas epithelial
differentiation of urogenital tract remains intact
(n = 4) (Figure 4, e and i)
. It is notable that
Shh and Gli2 mutant mice have blunted or missing
dorsal aspects of their most posterior aspect of cloaca
(n = 4) (Figure 4, e and i)
.
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Previously, we have shown that Gli2 and Gli3 possess overlapping functions in foregut development; Gli2-/-;Gli3+/- mice exhibit tracheoesophageal fistula and severe lung hypoplasia, and Gli2-/-;Gli3-/- mice do not develop any esophagus, lung, and trachea.43 To determine whether Gli2 and Gli3 possess overlapping functions in hindgut development, we examined the anorectal phenotype in Gli2;Gli3 mutant mice. Gli2+/-;Gli3+/- mice are viable and do not display any anorectal malformations. Gli2-/-;Gli3+/- and Gli2+/-;Gli3-/- mice can survive until birth, whereas most Gli2-/-;Gli3-/- mice die at E10.5 and only few of them develop up to E14.5.
All E18.5
Gli2-/-;Gli3+/-
(n = 8) and
Gli2+/-;Gli3-/-
mice (n = 8) displayed a complex and poorly
developed genital swelling in the perineal area (Figure 5; a, b, c, and d
). Although the external
appearance of perineal area and external genitalia was variable
depending on the sex and genotype, all double mutants exhibited a
persistent cloacal malformation. The perineal openings in all
Gli2-/-;Gli3+/-
and
Gli2+/-;Gli3-/-
mice were partially or fully covered by a bulging cloacal membrane
lined with uroepithelium (revealed at higher SEM magnifications; data
not shown). Histological sections of E14.5 mutant embryos confirmed
that all double mutants, including
Gli2-/-;Gli3-/-
mice (n = 2), develop a common outlet for
urinary and intestinal tracts (Figure 5; f, g, and h
). Persistent
cloaca is observed when either one dose of Gli3 is removed
in a Gli2-/- mutant background or one
dose of Gli2 is removed in a
Gli3-/- mutant background. The cloacal
phenotype of
Gli2-/-;Gli3+/-
and
Gli2+/-;Gli3-/-
mice is less severe than that of Shh null mice, and
Gli2-/-;Gli3-/-
mice have a severe cloacal phenotype (Figure 5h)
, which resembles that
of Shh null mice (Figure 2e)
. These observations illustrate that Gli2
and Gli3 possess both distinct and overlapping function in the
development of distal hindgut.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Moreover, gene dosage of
Gli2 and Gli3 was found to modulate the severity
of malformation suggesting that a precise degree of Shh signaling is
required for the normal development of rectum and anus.
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Biochemical and genetic studies have shown that the three mammalian Gli transcription factors (Gli1, Gli2, and Gli3) are involved in Shh signal transduction.39,40,43,45 Gli2 seems to be the major mediator of Shh signaling in vivo. In the developing foregut, Gli2 is required for the normal development of esophagus, trachea, and lung.27 Gli2-/- mice exhibit defects that are similar but milder than that observed in Shh null mice: stenosis of the esophagus and trachea, and lung lobulation defects.43 Gli3 plays a minor role in foregut development; Gli3-/- mice develop a normal trachea and esophagus, and only display subtle lung hypoplasia.55 Analogous to the foregut, Gli2 is important for the development of dorsal anorectum from the distal hindgut, whereas Gli3 mutant mice display anal stenosis, a less severe phenotype, which is the most common type of anorectal malformation in humans.2 In addition to their specific role in the development of distal hindgut, a gene dose-dependent effect observed in Gli2;Gli3 double mutant phenotypes suggest a conserved role of Shh signaling in both anterior and posterior ends of gut development.
Several theories on the embryogenesis of anorectal malformation have been presented in the past with considerable controversy. The first theory formulated by Tourneux49 and Retterer50 after examining embryos of sheep, rabbits, and pigs suggests that the crucial step in dividing the dorsal anorectum from the ventral urogenital tract is the formation of a septum. This remained the basis of our understanding until 1986 when more extensive anatomical and histological works refuted this theory because of the absence of morphological evidence to suggest septum formation either by the descent of a septum caudally or the formation and fusion of lateral ridges in the cloaca.18 More recently, studies of Sd mice noted that the most consistent finding in this animal model was the loss of the dorsal cloaca and the presence of a short cloacal membrane.16,56 In our anorectal mouse models, we found no evidence of active septation or lateral ridges formation/fusion on transverse sections of our mutant embryos, and similar to the Sd mice, we also observed a lack of formation of a normal dorsal cloaca. Our data now introduces a developmental model that furthers our understanding of anorectal malformation beyond the previous anatomical-based theories. Our data strongly suggest that morphogenetic events in the hindgut depend on a precise degree of Shh signal as demonstrated by the graded phenotypes seen with different gene dosages of Gli2 or Gli3. Furthermore, Shh signaling is likely to determine the spatial and temporal organization of tissue in the hindgut and aberrations in signaling is responsible for abnormal morphogenesis of the hindgut.
Mutant mice involving Shh signaling demonstrates a whole spectrum of anorectal malformations similar to those in humans and introduces, for the first time, a genetic basis for anorectal malformation. We conclude that Shh, Gli2, and Gli3 mutant mice are excellent animal models for studying the pathogenesis of anorectal malformations. The manipulation of the anatomical phenotype by molecular methods provides a powerful tool to rapidly expand our understanding of the morphogenesis of anorectal malformation.
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Acknowledgements |
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Footnotes |
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Supported by Canadian Institute of Health Research (to J. H. K.), the Heart and Stroke Foundation of Canada (grant no. T4607) (to P. K.), the National Cancer Institute of Canada (grant no. 9260) (to C.-c. H.), and the National Institutes of Health (grant HD37489) (to C.C.).
C.-c.H. is a Research Scientist of the NCIC supported with funds by the Canadian Cancer Society.
Accepted for publication April 27, 2001.
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