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Hoxd-13 expression in the development of hindgut in ethylenethiourea-exposed fetal rats
Journal of Pediatric Surgery (2010) 45, 755–761
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Hoxd-13 expression in the development of hindgut in ethylenethiourea-exposed fetal rats Zhang Dan, Zhang Zhi Bo, Zhang Tao, Zhang Shi Wei, Wang Da Jia, Zhang Shu Cheng, Yuan Zheng Wei, Wei-lin Wang ⁎ Department of Pediatric Surgery, The Second Affiliated Hospital, China Medical University, Shenyang 110004, PR China Received 7 May 2009; revised 12 November 2009; accepted 12 November 2010
Key words: Hoxd-13; Anorectal malformations; Hindgut development; Embryology; Cloaca; Rat
Abstract Purpose: Hoxd-13, as one of the most posterior genes among Hox genes, was reported to play a critical role in the development of the most posterior alimentary canal in vertebrates. This study investigated the expression pattern of Hoxd-13 in the hindgut development of the normal and ethylenethiourea (ETU)exposed rat embryos with anorectal malformations (ARMs) to find out the possible role of Hoxd-13 in the hindgut development and anorectal morphogenesis. Material and Method: The ETU murine model of ARMs was used via ETU 1% (125 mg/kg) on gestational day (gD) 10. Embryos were harvested via cesarean delivery on gD13 to gD21. Temporal and spatial expression of Hoxd-13 was evaluated in the normal fetal rats (n = 215) and ARMs rats (n = 218) using immunohistochemistry staining, reverse transcriptase polymerase chain reaction, and Western blot analysis. Results: Immunohistochemistry staining revealed that Hoxd-13 expression was confined to the epithelium of the hindgut, cloacal membrane, and urogenital sinus as well as the mesenchyme of the urorectal septum at all gestations in the normal group; however, in the ARMs group, the signal specific for Hoxd-13 was weak in the epithelium of the hindgut and cloacal membrane as well as the mesenchyme of the urorectal septum. Western blot analysis and reverse transcriptase polymerase chain reaction revealed that the level of Hoxd-13 expression was significantly decreased in the ARMs embryos compared with that in the normal embryos on gD13 to gD16 (P b .05) rather than on gD18 to gD21. Conclusions: The aberrations in spatiotemporal expression pattern of Hoxd-13 on gD13 to gD16 suggested that Hoxd-13 may be an essential inductive signal for normal development of the hindgut, and altered expression may contribute to the abnormal development of the hindgut and accordingly lead to ARMs. © 2010 Elsevier Inc. All rights reserved.
Hox genes encode transcription factors that play a fundamental role in patterning the anteroposterior axis of developing embryos [1,2]. There are 39 murine Hox genes that were divided into 4 linkage groups (A-D), each on a different chromosome. Hox genes have been numbered 1 to ⁎ Corresponding author. Fax: +86 24 23892617. E-mail address: [email protected] (W. Wang). 0022-3468/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2009.11.011
13 based on their positions within each cluster. All genes of a given murine cluster are transcribed from the same DNA strand and are expressed in successive, but partially overlapping, domains along the anteroposterior axis of the mouse embryo, according to their 3′ to 5′ order. Among the various sites of Hox genes expression, the alimentary canal is of particular interest because it represents one of the phylogenetically oldest structures. Hoxd-13, as one of the
756 most posterior genes in Hox genes, was reported to play a critical role in the development of the most posterior alimentary canal and urogenital duct in vertebrates [3]. Anorectal malformations (ARMs) are one of the most common abnormalities in the neonatal digestive system, which affect 1 in 4000 to 5000 newborns [4]. Although much work has been done to explore the pathogenesis and embryology of ARMs, the genetic basis remains unclear. It has been shown that Hoxd-13 gene is critical in the hindgut development, and Hoxd-13 mutant mice [3] or null mice [5] have been reported to show imperforate anus and other alterations of the posterior structures in varying degrees. The pattern of Hoxd-13 expression has been studied in several species. Hoxd-13 was strongly expressed in the mesenchyme of the genital tubercle of the mice [6], as well as in the hindgut and cloacal region [7], from which the terminal part of the intestine (rectum) and urogenital tracts develops. Hoxd-13 is expressed in the cognate zebra fish and chicken similarly in the most posterior restricted domain of the developing hindgut [8,9]. In the murine species, the expression of Hoxd-13 gene persists until perinatal stages [10,11]. Hoxd-13 was also observed to be expressed in the smooth muscle, anal sphincter, and levator ani muscle in the 10- to 15-week human embryos [12]. However, limited information is available about the role of Hoxd-13 in the development of the human gastrointestinal tract. This study investigated the expression pattern of Hoxd-13 in the hindgut development of the normal and ethylenethiourea (ETU)-exposed rat embryos with ARMs to determine the role of Hoxd-13 in the hindgut development and anorectal morphogenesis and further elucidate the genetic profile of this disease.
1. Material and methods 1.1. Animal model and tissue collection The research project was approved by the China Medical University Animal Ethics Committee. Seventy Wistar rats were time mated. The day on which sperm were found in a vaginal smear was considered as gestational day (gD) 0. The rats were kept individually in an air-conditioned, 12-hour light-dark cycle animal laboratory (The Key Laboratory of Health Ministry for Congenital Malformations) and fed with normal rat chow and tap water ad libitum. The ETU murine model of ARMs was used according to the procedure described previously [13,14]. One percent ETU water solution (ETU powder; s24419-426; SigmaAldrich) was kept in lightproof condition at 4°C. All rats were divided into 2 groups: ETU-exposed group and control group. Pregnant rats were gavage fed 125 mg/kg of 1% ETU on gD10; ETU-exposed group and control group were fed with equal dose of saline on gD10. One third of embryos per litter were harvested via cesarean delivery on gD13 to gD16 (the period of hindgut development) as well as on gD18 and gD21 (the term period
Z. Dan et al. of hindgut development) and then fixed in 4% paraformaldehyde/0.1 mol/L phosphate-buffered saline (PBS) for 12 to 24 hours according to their size. Afterward, the fixed embryos were embedded in paraffin followed by serial sagittal sectioning at 4-μm thickness. Some sections were examined for ARMs with hematoxylin and eosin staining. The morphogenesis of the hindgut, cloaca, rectum, notochord, and spinal cord was compared with age-matched controls. For Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR), the residual embryos per litter were frozen in liquid nitrogen for cryostat sections. Sagittal sequential sections were used to examine the position and morphous of the cloacal membrane, urorectal septum (URS), cloaca, hindgut, and tail groove according to the previous literature [13,14] to distinguish the occurrence of ARMs in ETU-exposed embryos before the microdissections. Afterward, the hindgut from gD13 to gD15 and the end segment of the rectum from gD16, gD18, and gD21 were dissected and removed free from surrounding tissues under magnification and immediately frozen in liquid nitrogen to prepare for Western blot analysis and RT-PCR. The abovementioned procedures were processed in 4°C cold room.
1.2. Immunohistochemistry staining Immunohistochemistry staining was performed as described previously [15]. Slides were pretreated with 3% H2O2 for 10 minutes at room temperature to quench endogenous peroxidase activity. Antigen retrieval was carried out by 10 minutes in 10 mmol/L citrate buffer (PH 6.0) at 100°C and 20 minutes at room temperature. The sections were washed 3 times with PBS for 5 minutes each and then incubated in 10% normal rabbit serum in PBS for 10 minutes. This was followed by incubation overnight at 4°C with primary Hoxd-13 antibody (1:150; sc-46364; Santa Cruz Biotechnology Inc, Santa Cruz, CA). Slides were then washed with PBS 3 times for 5 minutes each and incubated for 10 minutes with the secondary antibody, biotinylated rabbit antigoat immunoglobulin G (1:200 dilution). Sections were then treated with streptomycin anti-biotin peroxidase solution and diaminobenzidine tetrahydrochloride (DAB) substrate for detecting Hoxd-13–positive cells (Fu Zhou Mai Xin Biological Engineering Limited Company, Fu Zhou, China). Counterstaining was carried out with hematoxylin. Controls were routinely included. The specimens were photographed under the digitized microscope camera (Nikon E800, Japan). The positive results were determined by dark brown granules in the cytoplasm and calculated by 2 independent observers.
1.3. Western blot analysis Protein preparation was performed on approximately one half of the microdissected specimens as described previously [16]. Protein lysates were separated by electrophoresis using 12% sodium dodecyl sulfate–polyacrylamide gel, and
Hoxd-13 expression in ETU-exposed fetal rats proteins were subsequently transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). Western analysis used primary antibody at 1:200 for goat anti–HoxD13 (k-20, sc-46364, Santa Cruz Biotechnology Inc) and 1:2000 for donkey anti-goat horseradish peroxidase–conjugated secondary antibody (sc-2033, Santa Cruz Biotechnology Inc), each in 0.05% Tween-20 (p1379, Sigma-Aldrich Shanghai Trading Co.,Ltd) in PBS with 5% Carnation nonfat dry milk. Protein expression was visualized using Supersignal chemiluminescent substrate (Pierce).
1.4. Reverse transcriptase polymerase chain reaction Total RNA was extracted from microdissected samples by the RNeasy Mini Kit (Qiagen). The purity of the total RNA was measured as 260:280-nm ratio (expected values between 1.8 and 2.0). The RT-PCR experiments were performed using the One-Step RT-PCR kit (Invitrogen) with the following program: 50°C for 30 minutes and 35 cycles of 53.9°C, 1-minute annealing at 53.9°C, and 1-minute extension at 72°C. Each primer pair was designed to amplify products spanning multiple exons, thus distinguishing spliced messenger RNA (mRNA) from genomic DNA amplification. The RT-PCR primer pairs are listed below followed by the product size (base pairs [bp]): Hoxd-13: (f) 5′-CTA CAC GAG TCC CTA TCA GC-3′ and (r) 5′-CCG ACG GTA GAC GCA CAT-3′, 241 bp; β-actin: (f) 5′-GAT TGC CTC AGG ACA TTT CTG-3′ and (r) 5′-CTG GGT GTC CTG ATG TGC-3′, 690 bp. The densitometry of Hoxd-13 mRNA levels was normalized to that of β-actin, a housekeeping gene, and was expressed as a ratio.
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2.2. Immunohistochemistry staining results: spatiotemporal characteristics of Hoxd-13 distribution In the normal group, immunoreactivity specific to Hoxd13 was detected abundantly in the epithelium of the hindgut, cloacal membrane, and urogenital sinus (UGS) and the mesenchyme of the URS at all gestations. The appearance of Hoxd-13–positive cells increased progressively with advancing gestation. In the ARMs group, the abundant epithelial cells in the UGS showed positive staining at all gestations; and there was no difference in the expression pattern between the 2 groups, whereas a few positive cells were observed in the mesenchyme of the URS and the epithelium of the hindgut and cloacal membrane. However, the signal specific for Hoxd-13 was weak compared with those in the normal embryos at corresponding gestations. The comparison between the 2 groups on each gestational day was shown in Table 1.
2.3. Western blot analysis: time-dependent decrease of Hoxd-13 expression in ARMs embryos
All numerical data were presented as mean ± SD. Statistical analysis was performed using the 2-sample t test. P b .05 was considered as significance.
Hoxd-13 was detected as an approximately 36-kd band on Western blots of protein extracted from both normal and ARMs embryos (Fig. 4). The expression of Hoxd-13 protein was characterized by time-dependent changes. In the normal group, the expression of Hoxd-13 reached estimated optimal level; however, Hoxd-13 protein expression was faint in the ARMs group. The level of Hoxd-13 expression increased with advancing gestation in both groups. However, the level of Hoxd-13 protein was significantly decreased in the ARMs group compared with those in the normal embryos on gD13 to gD16 (156.9 ± 23.5 vs 102.7 ± 14.4, 165.5 ± 26.2 vs 104.5 ± 14.6, 169.6 ± 19.4 vs 108.2 ± 10.7, and 175.9 ± 20.5 vs 112.7 ± 15.4, respectively; P b .05). There was no significant decrease in the level on gD18 to gD21 (181.4 ± 16.4 vs 177.1 ± 10.7 and 183.9 ± 22.5 vs 179.7 ± 17.4, respectively).
2. Results
2.4. RT-PCR results: decreased transcriptional level of Hoxd-13 in ARMs embryos
1.5. Statistical analysis
2.1. Animal model In this study, there were 215 normal rat embryos and 218 ARMs embryos. None of the pregnant rats died after the ETU administration. In all ETU-exposed embryos, the length of the body was much shorter than that of the normal embryos; the tail was short or absent; externally visible spinal bifida and meningocele were observed as well. The incidence of ARMs in ETU-exposed group on E13 to E21 was 63.2% (218/345). The rectoprostatic urethral fistulas were the most frequent anomaly in male rats, whereas common cloaca was the top anomaly in female rats. In the normal group, there was no malformation; and the rectum and urethra were completely independent structures.
The RT-PCR amplification of Hoxd-13 in the normal embryos showed positive or strongly positive bands, whereas, in the ARMs group, it showed moderate or weak bands (Fig. 5). No amplified products were seen in the mock RT and PCR-negative samples. It demonstrated that Hoxd13 expression showed time-dependent changes in the developing hindgut. In the 2 groups, Hoxd-13 mRNA increased progressively with advancing gestation. However, the level of Hoxd-13 mRNA was lower in the ARMs embryos compared with that in the normal embryos at corresponding gestations. There was significant difference in the level of Hoxd-13 mRNA on gD13 to gD16 (0.35 ± 0.06 vs 0.13 ± 0.07, 0.51 ± 0.07 vs 0.35 ± 0.06, 0.62 ± 0.06 vs 0.40 ± 0.07, and 0.75 ± 0.05 vs 0.62 ± 0.05, respectively; P b
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Table 1
Hoxd-13 expression of embryos at different embryonic stages by the immunohistochemistry staining
Normal rat embryos gD13 Hoxd-13 expression occurred in the epithelium of the primary hindgut and URS. A few positive cells were scattered in the cloacal membrane. gD14 The URS was progressively descending, and the distance between the URS and cloacal membrane became smaller. Hoxd-13–positive cells were seen in the epithelium of the hindgut, URS, UGS, and the dorsal membrane (Fig. 1A). gD15 The epithelium in the top of URS fused with the epithelium of the dorsal cloacal membrane, and there was a thin anal membrane between the tail groove and the rectum. The most conspicuous reaction was localized in the anal membrane and the fusion zone (Fig. 2A). gD16 The anorectum was ultimately formed. Hoxd-13 immunoreactivity was observed in the epithelium of the URS, rectum, urethra, and bladder; in the anal canal; and in the mesenchyme of the URS (Fig. 3A). gD18 The abundant immunoreactivity for Hoxd-13 was localized in gD21 the epithelium and mesenchyme of the anorectum, bladder, and urethra.
.05) but not on gD18 to gD21 (1.07 ± 0.07 vs 0.89 ± 0.05 and 1.08 ± 0.05 vs 0.91 ± 0.07, respectively).
3. Discussion The previous work of our group and others has shown that a teratogenic dose of ETU produces ARMs in rat embryos [13,14]. This article investigated further the related protein
ARMs rat embryos The cloacal anal was wider than that in the normal embryos. Hoxd-13 expression occurred in the epithelium of the hindgut, cloacal membrane. The cloacal anal remained to have a wider appearance. A few positive cells were seen in the epithelium of the URS and cloacal membrane (Fig. 1B). The URS failed to fuse with the cloacal membrane. The cloacal anal remained as before. There were some positive cells in the epithelium of the URS, cloacal membrane, and hindgut (Fig. 2B).
The rectourethral fistula (or common cloaca) was observed. No Hoxd-13 staining was found in the mesenchyme of the URS and the epithelium of the fistula (Fig. 3B). The pattern of expression was similar to that in the normal group; in addition, some positive cells were seen in the epithelium of the fistula.
within ETU-produced ARMs embryos. Hoxd-13 is expressed in the developing hindgut of the normal as well as rat embryos with ARMs; however, the spatiotemporal expression pattern was impaired in ARMs group, suggesting that overdose of ETU disturbed Hoxd-13 expression in the hindgut development. The digestive and urogenital duct system of mammalian embryos initially shared a common posterior outlet, the cloaca, which was partitioned into a ventral primitive UGS and a dorsal rectum by the growth of the URS. In the early
Fig. 1 Immunohistochemical staining for Hoxd-13 in midsagittal sections of the murine embryos on gD14. A, In the normal group, the appearance of Hoxd-13–positive cells was seen in the epithelium of the hindgut, URS, and UGS as well as the mesenchyme of the URS. The epithelium of the dorsal membrane showed some positive staining. B, The positive staining was observed in the epithelium of the UGS of the ARMs embryos. Several positive cells occurred in the epithelium of the hindgut and cloacal membrane. The regions encircled by the red rectangle in the minipictures (original magnification ×40) were shown clearly by the magnified pictures (original magnification ×400). H indicates hindgut; CM, cloacal membrane; CL, cloaca.
Hoxd-13 expression in ETU-exposed fetal rats
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Fig. 2 Immunohistochemical study for Hoxd-13 in midsagittal sections of the murine embryos on gD15. A, In the normal group, the pattern of expression was similar with that on gD14 in the normal group. The most conspicuous reaction was localized in anal membrane and the fusion zone. B, The epithelium of the UGS showed positive staining in the ARMs group. Some positive staining was seen in the epithelium of the hindgut and cloacal membrane. The regions encircled by the red rectangle in the minipictures (original magnification ×40) were shown clearly by the magnified pictures (original magnification ×400). AM indicates anal membrane.
stage of the cloacal development, the main events were overgrowth of mesenchyme in the URS, adherence, confluence, and apoptosis of the epithelial cells in the cloaca, which lead the URS to elongate toward the dorsal end obviously [13]. The growth and expansion of the embryos contributed to reducing the distance between URS and cloacal membrane and the fusion of the 2 structures [17]. In this study, it was also observed that the URS fused with the cloacal membrane on gD15 in the normal embryos but not in the ARMs embryos, as previously shown by others. Whether the URS fuses with the cloacal membrane is one of the key arguments at present among various theories of ARMs
pathogenesis. It was suggested that the failure of the cloacal membrane to fuse with the URS was one of the etiologies of ARMs [13,14,18]. Accordingly, any factor that interferes with the fusion of the cloacal membrane and the URS is likely to result in the occurrence of ARMs. During the morphogenesis of the anorectum, there were numerous biological events involved, such as cell proliferation, differentiation, migration, and apoptosis, which were key events modulating the development of the hindgut. The failure of the URS to fuse with the cloacal membrane could reflect a decrease or arrest of cell proliferation. Recent studies have suggested that HOX genes were expressed in
Fig. 3 Immunohistochemical study for Hoxd-13 in midsagittal sections of the murine embryos on gD16. A, In the normal group, Hoxd-13 immunoreactivity was observed in the epithelium of the URS, rectum, urethra, and bladder; and some positive cells were seen in the mesenchyme of the URS and the epithelium of the anal canal. B, The rectourethral fistula was observed. No Hoxd-13 staining was found in the mesenchyme of the URS and the epithelium of the fistula. The regions encircled by the red rectangle in the minipictures (original magnification ×40) were shown clearly by the magnified pictures (original magnification ×400). F indicates rectourethral fistula.
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Fig. 4 Hoxd-13 protein was detected in the normal and ARMs developing hindgut tissue samples by Western blot analysis. It showed that they increased progressively with the gestations. On gD13 to gD16, the levels of Hoxd-13 protein in the ARMs embryos were significantly decreased than those in the normal group. On gD18 to gD21, there was no significant difference in HoxD-13 protein expression between the 2 groups. *Meets significance criteria (P value b .05).
the positionally restricted domain of the gut, which could induce cell proliferation and differentiation in the epithelium and mesenchyme [19,20]. The altered expression of Hoxd-13 in the mesenchyme and epithelium of the URS, hindgut, and
Z. Dan et al. cloacal membrane at the gD13 to gD16 stage is likely to hinder the proliferation, differentiation, and migration and disturb the normal apoptosis and cellular confluence, affecting the normal growth and expansion of the dorsal end and thus hindering the fusion of the URS and cloacal membrane. This study provided evidence of spatiotemporal difference in the expression pattern of Hoxd-13 in the hindgut development between the 2 groups and suggested that the difference of Hoxd-13 expression may be one of the factors that were responsible for the fusion failure of the cloacal membrane and the URS. In addition, in the current study, there was significant difference in the level of Hoxd-13 on gD13 to gD16 rather than on gD18 to gD21. The results were consistent with the concept that the gD13 to gD16 stage is a crucial period of the hindgut development [14]. In this period, the hindgut development changed dramatically from the initial structure with small capacity to the final rectum that communicated with the outside in the normal rat embryos. Any disorder occurring in this period may influence on the normal development of the hindgut. Thus, this period is the crucial period of teratogenesis. The aberrations of Hoxd-13 expression at the gD13 to gD16 stage may be one of the factors that affect the normal development of the hindgut and, accordingly, result in ARMs. The gD18 to gD21 stage lost the significant difference of Hoxd-13 expression pattern, which indicated that this stage might be a minor period for hindgut development. At present, ARMs have been considered as a multiplefactor–involved disease [16]. The anorectal development in the vertebrates resulting from the cooperation of endoderm,
Fig. 5 The RT-PCR analysis of Hoxd-13 mRNA expressed in the normal and ARMs developing hindgut tissue samples at each gestational day. It showed that the time-dependent expression of Hoxd-13 mRNA was characterized by increased levels from gD13 to gD21 in both groups and decreased significantly in ARMs embryos on gD13 to gD16 compared with that in the normal group on gD18 to gD21. *Meets significance criteria (P value b .05).
Hoxd-13 expression in ETU-exposed fetal rats mesoderm, and ectoderm needs abundant signal communications among 3 blastoderms. The transcription factor Hoxd-13 is one of the factors involved in the anorectal development. Sonic hedgehog (Shh) deriving from endoderm is regarded as the important signal implicated in the first phase of signaling from endoderm to mesoderm [16,19]. As such, it could be suggested that Shh could induce Hoxd-13 expression in the mesoderm, as Hoxd-13 expression levels are consistent with Shh expression and Hoxd-13 expression has been implicated as a reaction of the signal deriving from the endoderm [16]. It has also been implied that the fibroblast growth factor deriving from the mesoderm may be involved in the regulation of Hoxd-13 gene [21]. Hoxd-13 plays a role by regulating the downstream target genes, especially genes that directly assist in determining the cellular morphogenesis such as the cellular inherence and communication. It is reported that EphB2 and its ligand ephrinB2 play a key role in the cell-cell adhesion events at the extreme caudal midline. Salsi et al [22] found that Hoxd-13 gene directly controls the Eph expression in the limbs of Drosophila. Eph gene is considered as the immediate downstream gene of Hoxd-13 gene [23]. Interestingly, our previous data indicated that EphB2 receptor might be related to the development of ARMs [24]. In summary, the aberrations in spatiotemporal expression pattern of Hoxd-13 on gD13 to gD16, the crucial period of the hindgut development for the rat embryos, suggested that Hoxd-13 may be an essential inductive signal for normal development of the hindgut; and the altered expression may result from the disordered upstream signaling pathways and may contribute to the occurrence of ARMs by disturbing the expression pattern of the downstream signals. Further study on Hoxd-13 function and interactive actions in the anorectal development may facilitate understanding of the pathogenesis of ARMs.
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Hoxd-13 expression in the development of hindgut in ethylenethiourea-exposed fetal rats Zhang Dan, Zhang Zhi Bo, Zhang Tao, Zhang Shi Wei, Wang Da Jia, Zhang Shu Cheng, Yuan Zheng Wei, Wei-lin Wang ⁎ Department of Pediatric Surgery, The Second Affiliated Hospital, China Medical University, Shenyang 110004, PR China Received 7 May 2009; revised 12 November 2009; accepted 12 November 2010
Key words: Hoxd-13; Anorectal malformations; Hindgut development; Embryology; Cloaca; Rat
Abstract Purpose: Hoxd-13, as one of the most posterior genes among Hox genes, was reported to play a critical role in the development of the most posterior alimentary canal in vertebrates. This study investigated the expression pattern of Hoxd-13 in the hindgut development of the normal and ethylenethiourea (ETU)exposed rat embryos with anorectal malformations (ARMs) to find out the possible role of Hoxd-13 in the hindgut development and anorectal morphogenesis. Material and Method: The ETU murine model of ARMs was used via ETU 1% (125 mg/kg) on gestational day (gD) 10. Embryos were harvested via cesarean delivery on gD13 to gD21. Temporal and spatial expression of Hoxd-13 was evaluated in the normal fetal rats (n = 215) and ARMs rats (n = 218) using immunohistochemistry staining, reverse transcriptase polymerase chain reaction, and Western blot analysis. Results: Immunohistochemistry staining revealed that Hoxd-13 expression was confined to the epithelium of the hindgut, cloacal membrane, and urogenital sinus as well as the mesenchyme of the urorectal septum at all gestations in the normal group; however, in the ARMs group, the signal specific for Hoxd-13 was weak in the epithelium of the hindgut and cloacal membrane as well as the mesenchyme of the urorectal septum. Western blot analysis and reverse transcriptase polymerase chain reaction revealed that the level of Hoxd-13 expression was significantly decreased in the ARMs embryos compared with that in the normal embryos on gD13 to gD16 (P b .05) rather than on gD18 to gD21. Conclusions: The aberrations in spatiotemporal expression pattern of Hoxd-13 on gD13 to gD16 suggested that Hoxd-13 may be an essential inductive signal for normal development of the hindgut, and altered expression may contribute to the abnormal development of the hindgut and accordingly lead to ARMs. © 2010 Elsevier Inc. All rights reserved.
Hox genes encode transcription factors that play a fundamental role in patterning the anteroposterior axis of developing embryos [1,2]. There are 39 murine Hox genes that were divided into 4 linkage groups (A-D), each on a different chromosome. Hox genes have been numbered 1 to ⁎ Corresponding author. Fax: +86 24 23892617. E-mail address: [email protected] (W. Wang). 0022-3468/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2009.11.011
13 based on their positions within each cluster. All genes of a given murine cluster are transcribed from the same DNA strand and are expressed in successive, but partially overlapping, domains along the anteroposterior axis of the mouse embryo, according to their 3′ to 5′ order. Among the various sites of Hox genes expression, the alimentary canal is of particular interest because it represents one of the phylogenetically oldest structures. Hoxd-13, as one of the
756 most posterior genes in Hox genes, was reported to play a critical role in the development of the most posterior alimentary canal and urogenital duct in vertebrates [3]. Anorectal malformations (ARMs) are one of the most common abnormalities in the neonatal digestive system, which affect 1 in 4000 to 5000 newborns [4]. Although much work has been done to explore the pathogenesis and embryology of ARMs, the genetic basis remains unclear. It has been shown that Hoxd-13 gene is critical in the hindgut development, and Hoxd-13 mutant mice [3] or null mice [5] have been reported to show imperforate anus and other alterations of the posterior structures in varying degrees. The pattern of Hoxd-13 expression has been studied in several species. Hoxd-13 was strongly expressed in the mesenchyme of the genital tubercle of the mice [6], as well as in the hindgut and cloacal region [7], from which the terminal part of the intestine (rectum) and urogenital tracts develops. Hoxd-13 is expressed in the cognate zebra fish and chicken similarly in the most posterior restricted domain of the developing hindgut [8,9]. In the murine species, the expression of Hoxd-13 gene persists until perinatal stages [10,11]. Hoxd-13 was also observed to be expressed in the smooth muscle, anal sphincter, and levator ani muscle in the 10- to 15-week human embryos [12]. However, limited information is available about the role of Hoxd-13 in the development of the human gastrointestinal tract. This study investigated the expression pattern of Hoxd-13 in the hindgut development of the normal and ethylenethiourea (ETU)-exposed rat embryos with ARMs to determine the role of Hoxd-13 in the hindgut development and anorectal morphogenesis and further elucidate the genetic profile of this disease.
1. Material and methods 1.1. Animal model and tissue collection The research project was approved by the China Medical University Animal Ethics Committee. Seventy Wistar rats were time mated. The day on which sperm were found in a vaginal smear was considered as gestational day (gD) 0. The rats were kept individually in an air-conditioned, 12-hour light-dark cycle animal laboratory (The Key Laboratory of Health Ministry for Congenital Malformations) and fed with normal rat chow and tap water ad libitum. The ETU murine model of ARMs was used according to the procedure described previously [13,14]. One percent ETU water solution (ETU powder; s24419-426; SigmaAldrich) was kept in lightproof condition at 4°C. All rats were divided into 2 groups: ETU-exposed group and control group. Pregnant rats were gavage fed 125 mg/kg of 1% ETU on gD10; ETU-exposed group and control group were fed with equal dose of saline on gD10. One third of embryos per litter were harvested via cesarean delivery on gD13 to gD16 (the period of hindgut development) as well as on gD18 and gD21 (the term period
Z. Dan et al. of hindgut development) and then fixed in 4% paraformaldehyde/0.1 mol/L phosphate-buffered saline (PBS) for 12 to 24 hours according to their size. Afterward, the fixed embryos were embedded in paraffin followed by serial sagittal sectioning at 4-μm thickness. Some sections were examined for ARMs with hematoxylin and eosin staining. The morphogenesis of the hindgut, cloaca, rectum, notochord, and spinal cord was compared with age-matched controls. For Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR), the residual embryos per litter were frozen in liquid nitrogen for cryostat sections. Sagittal sequential sections were used to examine the position and morphous of the cloacal membrane, urorectal septum (URS), cloaca, hindgut, and tail groove according to the previous literature [13,14] to distinguish the occurrence of ARMs in ETU-exposed embryos before the microdissections. Afterward, the hindgut from gD13 to gD15 and the end segment of the rectum from gD16, gD18, and gD21 were dissected and removed free from surrounding tissues under magnification and immediately frozen in liquid nitrogen to prepare for Western blot analysis and RT-PCR. The abovementioned procedures were processed in 4°C cold room.
1.2. Immunohistochemistry staining Immunohistochemistry staining was performed as described previously [15]. Slides were pretreated with 3% H2O2 for 10 minutes at room temperature to quench endogenous peroxidase activity. Antigen retrieval was carried out by 10 minutes in 10 mmol/L citrate buffer (PH 6.0) at 100°C and 20 minutes at room temperature. The sections were washed 3 times with PBS for 5 minutes each and then incubated in 10% normal rabbit serum in PBS for 10 minutes. This was followed by incubation overnight at 4°C with primary Hoxd-13 antibody (1:150; sc-46364; Santa Cruz Biotechnology Inc, Santa Cruz, CA). Slides were then washed with PBS 3 times for 5 minutes each and incubated for 10 minutes with the secondary antibody, biotinylated rabbit antigoat immunoglobulin G (1:200 dilution). Sections were then treated with streptomycin anti-biotin peroxidase solution and diaminobenzidine tetrahydrochloride (DAB) substrate for detecting Hoxd-13–positive cells (Fu Zhou Mai Xin Biological Engineering Limited Company, Fu Zhou, China). Counterstaining was carried out with hematoxylin. Controls were routinely included. The specimens were photographed under the digitized microscope camera (Nikon E800, Japan). The positive results were determined by dark brown granules in the cytoplasm and calculated by 2 independent observers.
1.3. Western blot analysis Protein preparation was performed on approximately one half of the microdissected specimens as described previously [16]. Protein lysates were separated by electrophoresis using 12% sodium dodecyl sulfate–polyacrylamide gel, and
Hoxd-13 expression in ETU-exposed fetal rats proteins were subsequently transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). Western analysis used primary antibody at 1:200 for goat anti–HoxD13 (k-20, sc-46364, Santa Cruz Biotechnology Inc) and 1:2000 for donkey anti-goat horseradish peroxidase–conjugated secondary antibody (sc-2033, Santa Cruz Biotechnology Inc), each in 0.05% Tween-20 (p1379, Sigma-Aldrich Shanghai Trading Co.,Ltd) in PBS with 5% Carnation nonfat dry milk. Protein expression was visualized using Supersignal chemiluminescent substrate (Pierce).
1.4. Reverse transcriptase polymerase chain reaction Total RNA was extracted from microdissected samples by the RNeasy Mini Kit (Qiagen). The purity of the total RNA was measured as 260:280-nm ratio (expected values between 1.8 and 2.0). The RT-PCR experiments were performed using the One-Step RT-PCR kit (Invitrogen) with the following program: 50°C for 30 minutes and 35 cycles of 53.9°C, 1-minute annealing at 53.9°C, and 1-minute extension at 72°C. Each primer pair was designed to amplify products spanning multiple exons, thus distinguishing spliced messenger RNA (mRNA) from genomic DNA amplification. The RT-PCR primer pairs are listed below followed by the product size (base pairs [bp]): Hoxd-13: (f) 5′-CTA CAC GAG TCC CTA TCA GC-3′ and (r) 5′-CCG ACG GTA GAC GCA CAT-3′, 241 bp; β-actin: (f) 5′-GAT TGC CTC AGG ACA TTT CTG-3′ and (r) 5′-CTG GGT GTC CTG ATG TGC-3′, 690 bp. The densitometry of Hoxd-13 mRNA levels was normalized to that of β-actin, a housekeeping gene, and was expressed as a ratio.
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2.2. Immunohistochemistry staining results: spatiotemporal characteristics of Hoxd-13 distribution In the normal group, immunoreactivity specific to Hoxd13 was detected abundantly in the epithelium of the hindgut, cloacal membrane, and urogenital sinus (UGS) and the mesenchyme of the URS at all gestations. The appearance of Hoxd-13–positive cells increased progressively with advancing gestation. In the ARMs group, the abundant epithelial cells in the UGS showed positive staining at all gestations; and there was no difference in the expression pattern between the 2 groups, whereas a few positive cells were observed in the mesenchyme of the URS and the epithelium of the hindgut and cloacal membrane. However, the signal specific for Hoxd-13 was weak compared with those in the normal embryos at corresponding gestations. The comparison between the 2 groups on each gestational day was shown in Table 1.
2.3. Western blot analysis: time-dependent decrease of Hoxd-13 expression in ARMs embryos
All numerical data were presented as mean ± SD. Statistical analysis was performed using the 2-sample t test. P b .05 was considered as significance.
Hoxd-13 was detected as an approximately 36-kd band on Western blots of protein extracted from both normal and ARMs embryos (Fig. 4). The expression of Hoxd-13 protein was characterized by time-dependent changes. In the normal group, the expression of Hoxd-13 reached estimated optimal level; however, Hoxd-13 protein expression was faint in the ARMs group. The level of Hoxd-13 expression increased with advancing gestation in both groups. However, the level of Hoxd-13 protein was significantly decreased in the ARMs group compared with those in the normal embryos on gD13 to gD16 (156.9 ± 23.5 vs 102.7 ± 14.4, 165.5 ± 26.2 vs 104.5 ± 14.6, 169.6 ± 19.4 vs 108.2 ± 10.7, and 175.9 ± 20.5 vs 112.7 ± 15.4, respectively; P b .05). There was no significant decrease in the level on gD18 to gD21 (181.4 ± 16.4 vs 177.1 ± 10.7 and 183.9 ± 22.5 vs 179.7 ± 17.4, respectively).
2. Results
2.4. RT-PCR results: decreased transcriptional level of Hoxd-13 in ARMs embryos
1.5. Statistical analysis
2.1. Animal model In this study, there were 215 normal rat embryos and 218 ARMs embryos. None of the pregnant rats died after the ETU administration. In all ETU-exposed embryos, the length of the body was much shorter than that of the normal embryos; the tail was short or absent; externally visible spinal bifida and meningocele were observed as well. The incidence of ARMs in ETU-exposed group on E13 to E21 was 63.2% (218/345). The rectoprostatic urethral fistulas were the most frequent anomaly in male rats, whereas common cloaca was the top anomaly in female rats. In the normal group, there was no malformation; and the rectum and urethra were completely independent structures.
The RT-PCR amplification of Hoxd-13 in the normal embryos showed positive or strongly positive bands, whereas, in the ARMs group, it showed moderate or weak bands (Fig. 5). No amplified products were seen in the mock RT and PCR-negative samples. It demonstrated that Hoxd13 expression showed time-dependent changes in the developing hindgut. In the 2 groups, Hoxd-13 mRNA increased progressively with advancing gestation. However, the level of Hoxd-13 mRNA was lower in the ARMs embryos compared with that in the normal embryos at corresponding gestations. There was significant difference in the level of Hoxd-13 mRNA on gD13 to gD16 (0.35 ± 0.06 vs 0.13 ± 0.07, 0.51 ± 0.07 vs 0.35 ± 0.06, 0.62 ± 0.06 vs 0.40 ± 0.07, and 0.75 ± 0.05 vs 0.62 ± 0.05, respectively; P b
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Table 1
Hoxd-13 expression of embryos at different embryonic stages by the immunohistochemistry staining
Normal rat embryos gD13 Hoxd-13 expression occurred in the epithelium of the primary hindgut and URS. A few positive cells were scattered in the cloacal membrane. gD14 The URS was progressively descending, and the distance between the URS and cloacal membrane became smaller. Hoxd-13–positive cells were seen in the epithelium of the hindgut, URS, UGS, and the dorsal membrane (Fig. 1A). gD15 The epithelium in the top of URS fused with the epithelium of the dorsal cloacal membrane, and there was a thin anal membrane between the tail groove and the rectum. The most conspicuous reaction was localized in the anal membrane and the fusion zone (Fig. 2A). gD16 The anorectum was ultimately formed. Hoxd-13 immunoreactivity was observed in the epithelium of the URS, rectum, urethra, and bladder; in the anal canal; and in the mesenchyme of the URS (Fig. 3A). gD18 The abundant immunoreactivity for Hoxd-13 was localized in gD21 the epithelium and mesenchyme of the anorectum, bladder, and urethra.
.05) but not on gD18 to gD21 (1.07 ± 0.07 vs 0.89 ± 0.05 and 1.08 ± 0.05 vs 0.91 ± 0.07, respectively).
3. Discussion The previous work of our group and others has shown that a teratogenic dose of ETU produces ARMs in rat embryos [13,14]. This article investigated further the related protein
ARMs rat embryos The cloacal anal was wider than that in the normal embryos. Hoxd-13 expression occurred in the epithelium of the hindgut, cloacal membrane. The cloacal anal remained to have a wider appearance. A few positive cells were seen in the epithelium of the URS and cloacal membrane (Fig. 1B). The URS failed to fuse with the cloacal membrane. The cloacal anal remained as before. There were some positive cells in the epithelium of the URS, cloacal membrane, and hindgut (Fig. 2B).
The rectourethral fistula (or common cloaca) was observed. No Hoxd-13 staining was found in the mesenchyme of the URS and the epithelium of the fistula (Fig. 3B). The pattern of expression was similar to that in the normal group; in addition, some positive cells were seen in the epithelium of the fistula.
within ETU-produced ARMs embryos. Hoxd-13 is expressed in the developing hindgut of the normal as well as rat embryos with ARMs; however, the spatiotemporal expression pattern was impaired in ARMs group, suggesting that overdose of ETU disturbed Hoxd-13 expression in the hindgut development. The digestive and urogenital duct system of mammalian embryos initially shared a common posterior outlet, the cloaca, which was partitioned into a ventral primitive UGS and a dorsal rectum by the growth of the URS. In the early
Fig. 1 Immunohistochemical staining for Hoxd-13 in midsagittal sections of the murine embryos on gD14. A, In the normal group, the appearance of Hoxd-13–positive cells was seen in the epithelium of the hindgut, URS, and UGS as well as the mesenchyme of the URS. The epithelium of the dorsal membrane showed some positive staining. B, The positive staining was observed in the epithelium of the UGS of the ARMs embryos. Several positive cells occurred in the epithelium of the hindgut and cloacal membrane. The regions encircled by the red rectangle in the minipictures (original magnification ×40) were shown clearly by the magnified pictures (original magnification ×400). H indicates hindgut; CM, cloacal membrane; CL, cloaca.
Hoxd-13 expression in ETU-exposed fetal rats
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Fig. 2 Immunohistochemical study for Hoxd-13 in midsagittal sections of the murine embryos on gD15. A, In the normal group, the pattern of expression was similar with that on gD14 in the normal group. The most conspicuous reaction was localized in anal membrane and the fusion zone. B, The epithelium of the UGS showed positive staining in the ARMs group. Some positive staining was seen in the epithelium of the hindgut and cloacal membrane. The regions encircled by the red rectangle in the minipictures (original magnification ×40) were shown clearly by the magnified pictures (original magnification ×400). AM indicates anal membrane.
stage of the cloacal development, the main events were overgrowth of mesenchyme in the URS, adherence, confluence, and apoptosis of the epithelial cells in the cloaca, which lead the URS to elongate toward the dorsal end obviously [13]. The growth and expansion of the embryos contributed to reducing the distance between URS and cloacal membrane and the fusion of the 2 structures [17]. In this study, it was also observed that the URS fused with the cloacal membrane on gD15 in the normal embryos but not in the ARMs embryos, as previously shown by others. Whether the URS fuses with the cloacal membrane is one of the key arguments at present among various theories of ARMs
pathogenesis. It was suggested that the failure of the cloacal membrane to fuse with the URS was one of the etiologies of ARMs [13,14,18]. Accordingly, any factor that interferes with the fusion of the cloacal membrane and the URS is likely to result in the occurrence of ARMs. During the morphogenesis of the anorectum, there were numerous biological events involved, such as cell proliferation, differentiation, migration, and apoptosis, which were key events modulating the development of the hindgut. The failure of the URS to fuse with the cloacal membrane could reflect a decrease or arrest of cell proliferation. Recent studies have suggested that HOX genes were expressed in
Fig. 3 Immunohistochemical study for Hoxd-13 in midsagittal sections of the murine embryos on gD16. A, In the normal group, Hoxd-13 immunoreactivity was observed in the epithelium of the URS, rectum, urethra, and bladder; and some positive cells were seen in the mesenchyme of the URS and the epithelium of the anal canal. B, The rectourethral fistula was observed. No Hoxd-13 staining was found in the mesenchyme of the URS and the epithelium of the fistula. The regions encircled by the red rectangle in the minipictures (original magnification ×40) were shown clearly by the magnified pictures (original magnification ×400). F indicates rectourethral fistula.
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Fig. 4 Hoxd-13 protein was detected in the normal and ARMs developing hindgut tissue samples by Western blot analysis. It showed that they increased progressively with the gestations. On gD13 to gD16, the levels of Hoxd-13 protein in the ARMs embryos were significantly decreased than those in the normal group. On gD18 to gD21, there was no significant difference in HoxD-13 protein expression between the 2 groups. *Meets significance criteria (P value b .05).
the positionally restricted domain of the gut, which could induce cell proliferation and differentiation in the epithelium and mesenchyme [19,20]. The altered expression of Hoxd-13 in the mesenchyme and epithelium of the URS, hindgut, and
Z. Dan et al. cloacal membrane at the gD13 to gD16 stage is likely to hinder the proliferation, differentiation, and migration and disturb the normal apoptosis and cellular confluence, affecting the normal growth and expansion of the dorsal end and thus hindering the fusion of the URS and cloacal membrane. This study provided evidence of spatiotemporal difference in the expression pattern of Hoxd-13 in the hindgut development between the 2 groups and suggested that the difference of Hoxd-13 expression may be one of the factors that were responsible for the fusion failure of the cloacal membrane and the URS. In addition, in the current study, there was significant difference in the level of Hoxd-13 on gD13 to gD16 rather than on gD18 to gD21. The results were consistent with the concept that the gD13 to gD16 stage is a crucial period of the hindgut development [14]. In this period, the hindgut development changed dramatically from the initial structure with small capacity to the final rectum that communicated with the outside in the normal rat embryos. Any disorder occurring in this period may influence on the normal development of the hindgut. Thus, this period is the crucial period of teratogenesis. The aberrations of Hoxd-13 expression at the gD13 to gD16 stage may be one of the factors that affect the normal development of the hindgut and, accordingly, result in ARMs. The gD18 to gD21 stage lost the significant difference of Hoxd-13 expression pattern, which indicated that this stage might be a minor period for hindgut development. At present, ARMs have been considered as a multiplefactor–involved disease [16]. The anorectal development in the vertebrates resulting from the cooperation of endoderm,
Fig. 5 The RT-PCR analysis of Hoxd-13 mRNA expressed in the normal and ARMs developing hindgut tissue samples at each gestational day. It showed that the time-dependent expression of Hoxd-13 mRNA was characterized by increased levels from gD13 to gD21 in both groups and decreased significantly in ARMs embryos on gD13 to gD16 compared with that in the normal group on gD18 to gD21. *Meets significance criteria (P value b .05).
Hoxd-13 expression in ETU-exposed fetal rats mesoderm, and ectoderm needs abundant signal communications among 3 blastoderms. The transcription factor Hoxd-13 is one of the factors involved in the anorectal development. Sonic hedgehog (Shh) deriving from endoderm is regarded as the important signal implicated in the first phase of signaling from endoderm to mesoderm [16,19]. As such, it could be suggested that Shh could induce Hoxd-13 expression in the mesoderm, as Hoxd-13 expression levels are consistent with Shh expression and Hoxd-13 expression has been implicated as a reaction of the signal deriving from the endoderm [16]. It has also been implied that the fibroblast growth factor deriving from the mesoderm may be involved in the regulation of Hoxd-13 gene [21]. Hoxd-13 plays a role by regulating the downstream target genes, especially genes that directly assist in determining the cellular morphogenesis such as the cellular inherence and communication. It is reported that EphB2 and its ligand ephrinB2 play a key role in the cell-cell adhesion events at the extreme caudal midline. Salsi et al [22] found that Hoxd-13 gene directly controls the Eph expression in the limbs of Drosophila. Eph gene is considered as the immediate downstream gene of Hoxd-13 gene [23]. Interestingly, our previous data indicated that EphB2 receptor might be related to the development of ARMs [24]. In summary, the aberrations in spatiotemporal expression pattern of Hoxd-13 on gD13 to gD16, the crucial period of the hindgut development for the rat embryos, suggested that Hoxd-13 may be an essential inductive signal for normal development of the hindgut; and the altered expression may result from the disordered upstream signaling pathways and may contribute to the occurrence of ARMs by disturbing the expression pattern of the downstream signals. Further study on Hoxd-13 function and interactive actions in the anorectal development may facilitate understanding of the pathogenesis of ARMs.
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