Anorectal Malformations Caused by Defects in Sonic Hedgehog Signaling
2001; Elsevier BV; Volume: 159; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)61747-6
ISSN1525-2191
AutoresRong Mo, Jae Hong Kim, Jianrong Zhang, Chin Chiang, Chi‐chung Hui, Peter C.W. Kim,
Tópico(s)Teratomas and Epidermoid Cysts
ResumoAnorectal malformations are a common clinical problem affecting the development of the distal hindgut in infants. The spectrum of anorectal malformations ranges from the mildly stenotic anus to imperforate anus with a fistula between the urinary and intestinal tracts to the most severe form, persistent cloaca. The etiology, embryology, and pathogenesis of anorectal malformations are poorly understood and controversial. Sonic hedgehog (Shh) is an endoderm-derived signaling molecule that induces mesodermal gene expression in the chick hindgut. However, the role of Shh signaling in mammalian hindgut development is unknown. Here, we show that mutant mice with various defects in the Shh signaling pathway exhibit a spectrum of distal hindgut defects mimicking human anorectal malformations. Shh null-mutant mice display persistent cloaca. Mutant mice lacking Gli2 or Gli3, two zinc finger transcription factors involved in Shh signaling, respectively, exhibit imperforate anus with recto-urethral fistula and anal stenosis. Furthermore, persistent cloaca is also observed in Gli2−/−;Gli3+/−, Gli2+/−;Gli3−/−, and Gli2−/−;Gli3−/− mice demonstrating a gene dose-dependent effect. Therefore, Shh signaling is essential for normal development of the distal hindgut in mice and mutations affecting Shh signaling produce a spectrum of anorectal malformations that may reveal new insights into their human disease equivalents. Anorectal malformations are a common clinical problem affecting the development of the distal hindgut in infants. The spectrum of anorectal malformations ranges from the mildly stenotic anus to imperforate anus with a fistula between the urinary and intestinal tracts to the most severe form, persistent cloaca. The etiology, embryology, and pathogenesis of anorectal malformations are poorly understood and controversial. Sonic hedgehog (Shh) is an endoderm-derived signaling molecule that induces mesodermal gene expression in the chick hindgut. However, the role of Shh signaling in mammalian hindgut development is unknown. Here, we show that mutant mice with various defects in the Shh signaling pathway exhibit a spectrum of distal hindgut defects mimicking human anorectal malformations. Shh null-mutant mice display persistent cloaca. Mutant mice lacking Gli2 or Gli3, two zinc finger transcription factors involved in Shh signaling, respectively, exhibit imperforate anus with recto-urethral fistula and anal stenosis. Furthermore, persistent cloaca is also observed in Gli2−/−;Gli3+/−, Gli2+/−;Gli3−/−, and Gli2−/−;Gli3−/− mice demonstrating a gene dose-dependent effect. Therefore, Shh signaling is essential for normal development of the distal hindgut in mice and mutations affecting Shh signaling produce a spectrum of anorectal malformations that may reveal new insights into their human disease equivalents. Anorectal malformations encompass a broad spectrum of congenital defects that frequently necessitates urgent surgical treatment in the newborn period, most often because of intestinal obstruction and sepsis. One of the most common anomalies, imperforate anus, has an incidence of 1 in 5000 and carries with it significant chronic morbidity, particularly with fecal incontinence. The most severe form of anorectal malformation, the cloacae, where distal intestinal and genitourinary tracts remain in a common channel, is much less common than imperforate anus (1 in 50,000) but has more significant serious long-term medical problems including gender assignment.1Stevenson R Human Malformations and Related Anomalies.in: Stevenson R Hall JG Goodman RM Oxford University Press, London1993: 493-499Google Scholar, 2Skandalakis JE Gray SW Ricketts R The colon and rectum.in: Skandalakis J Gray SW Embryology for Surgeons. 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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.34Roberts DJ Johnson RL Burke AC Nelson CE Morgan BA Tabin C Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut.Development. 1995; 121: 3163-3174Crossref PubMed Google Scholar 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 Gli3.29Chiang C Litingtung Y Lee E Young KE Corden JL Westphal H Beachy PA Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.Nature. 1996; 383: 407-413Crossref PubMed Scopus (2545) Google Scholar, 40Mo R Freer AM Zinyk DL Crackower MA Michaud J Heng HH Chik KW Shi XM Tsui LC Cheng SH Joyner AL Hui C Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development.Development. 1997; 124: 113-123Crossref PubMed Google Scholar, 41Hui CC Joyner AL A mouse model of Greig cephalopolysyndactyly syndrome: the extra-toesJ mutation contains an intragenic deletion of the Gli3 gene.Nat Genet. 1993; 3: 241-246Crossref PubMed Scopus (609) Google Scholar, 42Kimmel SG Mo R Hui CC Kim PC New mouse models of congenital anorectal malformations.J Pediatr Surg. 2000; 35: 227-230Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar 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. Gli2 mutant mice carry a targeted deletion of the DNA-binding zinc-finger motifs of the gene.24Ming JE Roessler E Muenke M Human developmental disorders and the Sonic hedgehog pathway.Mol Med Today. 1998; 4: 343-349Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar Gli3 mutant mice are spontaneous null mutants with a large 3′ deletion of the gene.25Goodrich LV Scott MP Hedgehog and patched in neural development and disease.Neuron. 1998; 21: 1243-1257Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar Gli2+/−, Gli3+/−, and Gli2+/−;Gli3+/− mice were maintained in a mixed 129 and CD1 background. 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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. 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. Dissected embryos were fixed in osmium tetroxide, dehydrated in ethanol, mounted with carbon paint, and examined on JEOL820 SEM as described.44Malick LE Wison RB Scanning Electron Microscopy.in: Johari O IIT Research Institute, Chicago1975: 259-266Google Scholar Whole-mount in situ hybridization was performed according to the standard protocol.43Motoyama J Liu J Mo R Ding Q Post M Hui CC Essential function of Gli2 and Gli3 in the formation of lung, trachea and oesophagus.Nat Genet. 1998; 20: 54-57Crossref PubMed Scopus (458) Google Scholar, 45Ding Q Motoyama J Gasca S et al.Diminished Sonic hedgehog signaling and lack of floor plate differentiation in Gli2 mutant mice.Development. 1998; 125: 2533-2543Crossref PubMed Google Scholar, 46Conlon RA Herrmann BG Detection of messenger RNA by in situ hybridization to postimplantation embryo whole mounts.Methods Enzymol. 1993; 225: 373-383Crossref PubMed Scopus (83) Google Scholar Section in situ was performed according to published procedures.41Hui CC Joyner AL A mouse model of Greig cephalopolysyndactyly syndrome: the extra-toesJ mutation contains an intragenic deletion of the Gli3 gene.Nat Genet. 1993; 3: 241-246Crossref PubMed Scopus (609) Google Scholar, 47Schaeren-Wiemers N Gerfin-Moser A A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes.Histochemistry. 1993; 100: 431-440Crossref PubMed Scopus (1084) Google Scholar The probes used were Gli1, Gli2, and Gli3.48Hui CC Slusarski D Platt KA Holmgren R Joyner AL Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm- and mesoderm-derived tissues suggests multiple roles during postimplantation development.Dev Biol. 1994; 162: 402-413Crossref PubMed Scopus (411) Google Scholar 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).Figure 2Cloacal malformation in Shh−/− mice. SEM of E18.5 wild-type and Shh−/− mice showing the normal female genitalia of E18.5 wild-type mice (a) as compared to the complex cloacal opening with poorly developed external genitalia in the perineum of E18.5 Shh−/−mice (b) and a 22-week aborted human fetus (c). In b, the bulge in the cloaca was lined with uroepithelium as revealed by higher magnification (data not shown). In c, the boxed area indicates the partially covered cloacal opening. In E14.5 wild-type embryo (d), bladder and urethra are well separated from the anorectum. In E14.5 Shh−/− embryos, distal intestinal tract and ureters are connected to a common cloacal cavity (e) similar to en bloc dissected specimen from the aborted human embryo (f), which demonstrates ureters and distal intestinal tract (metal probes in place) draining into the cloacal cavity. Abbreviations: b, bladder; c, cloaca; ft, fallopian tube; gt, genital tubercle; gs, genital swelling; hg, hindgut; k, kidney; r, rectum; u, urethra; ur, ureter.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Imperforate anus in Gli2−/− mice and anal stenosis in Gli3−/− mice. The SEM of genital and anorectal regions of E18.5 wild-type (a), Gli2−/−(b), and Gli3−/−(c) mice. Large and small arrowheads indicate urethral and future vaginal openings, respectively, and arrows represent anal orifices (a and c). Gli2−/− mice display a single urethral opening in the perineum (indicated by the white arrowhead in b), whereas Gli3−/− mice exhibit anal stenosis (c). Sagittal sections reveal the absence of anus with a fistula (indicated by arrow) between distal intestinal tract and proximal urethra in Gli2−/−mice (e), and a narrowed anus (indicated by arrow) with normal lower urinary tract and rectum in Gli3−/− mice (f). Injection of radiographic contrast material into distal intestinal tract demonstrates a communication between the intestinal and urinary tracts in Gli2−/− mice (h), whereas a simple outline of the anus and rectum is revealed in the wild-type animal (g). En bloc microdissection of the embryos reveals that the distal intestine is connected to the lower urinary tract in Gli2−/− mice (h), and a narrowed anus with wedged meconium demonstrating anal stenosis in Gli3−/− mice (i). The lower urinary and intestinal tracts are outlined by white dots. Abbreviations: a, anus; b, bladder; c, cloaca; gt, genital tubercle; r, rectum; u, urethra.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 poorl
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