The Expression Of Shh/BMP4 Signaling Pathway In The Anorectal Malformations

IntroductionCongenital anorectal malformations(ARMs) comprise a wide spectrum of diseases,which resulted from impeded hindgut development during embryonic stage, and involve the anus and rectum as well as the urogenital tracts.ARMs are the most common alimentary deformities and severely influence life quality of the patients. Some problems such as constipation and incontinence remain even after operation.Pathogenesis of ARMs is too complex to be well understood.The abnormal regulations of several developmental genes have been implicated in the development of ARMs.However,the pathogenic genes of ARMs are still unlocated.Therefore,it is significant to investigate pathogenesis of ARMs on molecular level.Recent study has demonstrated that Shh/BMP4 signaling pathway is involved in the morphogenesis of many organ systems during embryonic development in higher vertebrates.In the gut development,Shh/BMP4 signaling pathway also effects on several aspects including the epithelial proliferation,the mesenchymal stratification,region-specific differentiation of the gut.Recent study in the human digestive tract indicated that mutations in members of the Shh/BMP4 signaling pathway are associated with different malformations such as imperforate anus,esophageal atresia /tracheoesophageal fistula,juvenile polyposis syndrome and etc.Experimental studies on Shh cascade mutant mice showed the mice develop imperforate anus that resemble humans and vary in severity from stenotic anus to complex cloacal malformations.On the basis of these findings,we hypothesized that the Shh/BMP4 pathway plays a role in the development of the ARMs.This study includes:â‘ The embryomorphogenesis and expression of Shh/BMP4 signaling in rat embryos with ARMs induced by ETU:Rat embryos with ARMs were obtained by treating with administration of ethylenethiourea(ETU) on pregnant rats. The cloacal morphogenesis and the expression patterns and levels of Shh,Gli2 and BMP4,were continuously and dynamicly studied by immunohistochemistry,RT-PCR and Western-blotting to explore the mechanism of ARMs.â‘¡The expression of Shh/BMP signaling in human ARMs:Shh,Gli2 and BMP4 expression in terminal rectum of the patients with ARMs and control groups were studied to explore the relativity of ARMs to Shh/BMP4 signaling pathway and to investigate pathogenic genes.Materials and MethodsMaterials1.Animals:Wistar rats were provided by Medical Animal Center in the Shengjing Hospital of China Medical University.2.Samples:Among 40 cases with anorectal malformations(15 high type and 25 low type) operated in our department,27 were males,and 13 were females.The average age was 1.5 months.10 normal controls were obtained from the bodies died of neonatal asphyxia,and average aged 1.0 month.Tissues were removed from terminal pouch of rectum and stored immediately in -75℃refrigerator.3.Reagents and instruments:â‘ Major reagents:ETU(2-Imidazolidinethione,98%;Aldrich Chemical Co,Inc, USA).The The specific antibodies to Shh,Gli2 and BMP4 were bought from the Santa Cruz biological company.The SP immunohistochemistry kit was bought from the Maxin biological technique Ltd.in Fuzhou.RT-PCR Kit,Trizol(mRNA extraction system) were provided by TaKaBa Biotechnology(Dalian) Co.,Ltd.The primers for Shh,Gli2,BMP4 andβ-actin were made by Invitrogen Biotechnology Co.,Ltd. â‘¡Major instruments:Nikon Eclipse E₈₀₀ opticmicroscope;PTC-200^TM PCR extender;The image collect system is NIS-Elements F_2.30.Methods1.The embryonic processes and expression of Shh/BMP4 signaling in rat embryos with ARMs induced by ETU:â‘ Animal modle:The timed-pregnant rats were divided into two groups(the ETU treated group and the control group) in random.The ETU treated group females were gavage-fed with 1%ETU at 125 mg/kg b.w.on gestational days10(gD10).The control group received the same volume of Sodium Chloride on the same day.Embryos were harvested via caesarean section on gD12.5, 13.5,14,15 and gD16,respectively.â‘¡Immunohistochemistry staining:The whole embryos from 3 ETU treated females and 3 control females from gD12.5 to 16 were fixed in 4%formaldehyde for 24 hour,processed,embedded in paraffin wax and serially sliced at 4μm sagittally.The slices were stained with immunohistochemistry staining and were examined microscopically.â‘¢RT-PCR:We determined the expression levels of Shh,Gli2 and BMP4 mRNA in the cloaca of the embryos from 2 ETU treated females and 2 control females at different time point.â‘£Western blotting: The fetuses on gD16 harvested from the control group and the ETU treated group were divided into three groups:the control group,the embryos without ARMs treated by ETU and the ARMs embryos treated by ETU.The protein expression of Shh,Gli2 and BMP4 in the rectum of the fetuses were detected and analyzed.2.The expression of Shh/BMP4 signaling in human ARMs:The expression patterns and levels of Shh,Gli2 and BMP4 genes in rectal samples of 40 cases with ARMs(15 high type and 25 low type) and 10 normal controls were studied by immunohistochemistry staining,RT-PCR and Western blotting.3.Statistical Analysis:All values are expressed as(?)±s,and the data were analyzed by One-way ANOVA or by t-test with SPSS13.0 for windows software,with P＜0.05 considered to indicated statistical significance. Results1.The embryonic processes and expression of Shh/BMP4 signaling in rat embryos with ARMs induced by ETU.Immunohistochemistry results:The embryos administered ETU showed high occurrence(63.2%) of ARMs and may used as a stable and reliable ARMs animal models.Histological observation of control embryos represented as follows:During normal cloacal developmental processes in rats,before gD 14,the genital tubercle developed and grew substantially,and the cloaca shifted ventrocaudally according to genital tubercle development.The cloaca configuration changes and obviously is divided into ventral urogenital sinus and dorsal primitive rectum.At the same time; from gD14 to gD16,urorectal septum descended with its distance to the cloacal membrane decreasing gradually,and fused with the cloacal membrane.Characters of cloaca development in ETU ARMs rat embryos were as follows:A.Cloacal configuration was abnormal.B.Urorectal septum descended incompletely,and never fused with cloaca membrane.In normal controls,immunoreactivity specific to Shh and Gli2 was detected most abundantly in the epithelium of the cloaca,urinary bladder, urethra,rectum and anus.The immunoreactivity for BMP4 was localized in the mesodermal cells and the mesenchyme beneath the epithelium of the cloaca and rectum and in urorectal septum.The most staining intensity of BMP4 occurred in the urorectal septum and in the mesenchyme surrounding the rectum on gD14.In ETU-treated embryos,the immunoreactivity specific to Shh,Gli2 and BMP4 were remarkably weak compared with those in the normal controls.From these histologic findings,teratogenic doses of ETU remarkably disturbed Shh/BMP4 signaling expression in the cloacal regions.RT-PCR results:The PCR experiment successfully yielded the amplified fragments of expected size forβ-actin(690 bp),Shh(187 bp),Gli2(158bp) and BMP4 (493bp) in each sample.Intensities of the bands corresponding toβ-actin were similar among all the samples.This confirmed that Shh/BMP4 expressed in control and experimental samples throughout the cloaca developmental period(gD12.5-16). Semiquantitative results demonstrated that the top of Shh,Gli2 and BMP4 mRNA expression curve was on gD12.5-14,and Shh/BMP4 expression levels were different in the developing cloaca of ETU-treated rat embryos on gD12.5-16 from those of the control embryos.Shh and Gli2 expression levels were significantly decreased on gD12.5-14(BMP4 on gD14-16) in developing cloaca of ETU-treated rat embryos as compared to the controls(P＜0.05).Western blotting results:Shh,Gli2 and BMP4 protein expression were evident in the rectums harvested from the control embryos and the embryos without ARMs treated by ETU on gD16.Shh,Gli2 and BMP4 expression levels(1.070±0.19, 1.490±0.05 and 0.926±0.15) of the embryos without ARMs treated by ETU diminished but did not reach the significant levels compared with controls(1.127±0.13, 1.527±0.16 and 0.980±0.05)(P＞0.05).Immunoblotting detected low levels of Shh/BMP4 protein in rectal tissues collected from the ARMs group treated by ETU. There were significant difference between the ARMs group and the embryos without ARMs treated by ETU(0.886±0.09,1.060±0.08 and 0.913±0.04) and the control group and the embryos without ARMs treated by ETU(P＜0.05).2.Expression of Shh/BMP4 signaling in human samples.Immunohistoc(?)emistry results:There were obvious immunostaining of Shh and Gli2 showed in the epithelium of the rectum,and positive staining for BMP4 showed in the mesenchyme and in the mesodermal cells underlying the epithelium and within the muscular layers of the gut.In the high ARMs,however,Shh,Gli2 and BMP4 expression were weaker than those in the control and low ARMs.RT-PCR results:We found that expression levels of Shh,Gli2 and BMP4 mRNA in terminal rectum of high ARMs(0.63±0.27,0.23±0.08 and 0.69±0.25) was significantly lower than those in low ARMs(0.89±0.39,0.91±0.32 and 0.98±0.33) and in normal rectum(0.91±0.71,1.24±0.38 and 1.11±0.45)(P＜0.05).However,there was no significant difference between the low ARMs group and the control group (except Gli2mRNA)(P＞0.05).Western blotting results:Average gray value(G-value) represented the level of protein expression of Shh,Gli2 and BMP4.G-values of Shh,Gli2 and BMP4 in the high ARMs(0.920±0.09,1.106±0.11 and 1.070±0.10) were significantly lower than in the low ARMs(1.317±0.13,1.210±0.10 and 1.137±0.14 ) and the control(1.586±0.08, 1.430±0.09 and 1.520±0.08)(P＜0.05).However,there was no significant difference between the low ARMs group and the control group in the expression levels of these genes.Conclusions1.This study showed that Shh/BMP4 was expressed in the developing cloaca of normal rat embryos,and morphogenic events in the cloaca were relative to Shh signal induction.2.Expression levels of Shh/BMP4 signaling were reduced in the cloaca of ETU-treated embryos.It suggested that ETU affected the expression of Shh/BMP4.3.During the critical stages of the clocal development,the aberrations in Shh/BMP4 signaling may be responsible for the abnormal morphogenesis of the cloaca. We presumed the reduced level of Shh/BMP4 expression may contribute to the ARMs.4.Shh/BMP4 signaling expressed in the terminal pouch of the human rectum and regulated the normal anorectal development.5.The expression level of Shh/BMP4 signaling in high ARMs was lower than that of the control group.Down-regulation of Shh/BMP4 signaling pathway was closely relative to the occurrence of high ARMs.6.The expression pattern of Shh/BMP4 signaling of low ARMs was normal.The result suggested there were still some other mechanisms responsible for the occurrence of low ARMs.