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Sonic hedgehog expression in the development of hindgut in ETU-exposed fetal rats

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Abstract

Sonic hedgehog (Shh) has been shown to be involved in the morphogenesis of many organ systems including the notochord, floor plate and limbs, as well as in the development of the left–right axis in vertebrates. Recent evidence suggests the Shh cascade plays a crucial role in the development of the foregut and hindgut. We have previously shown that prenatal exposure of fetal rats to ethylenethiourea (ETU) induces hindgut malformations and other abnormalities of the VACTERL association. The aim of this study was to determine the pattern of expression of Shh and its downstream genes during hindgut development in ETU-exposed embryos with anorectal malformations (ARMs). Pregnant Sprague–Dawley rats were mated together overnight and a positive vaginal plug was marked as gD0. On gD10, 1% ETU (125 mg/kg) was given to the experimental group and controls received the same volume of saline. Embryos were collected from both groups at gD12–16. The developing hindgut of each embryo was dissected under magnification and snap frozen. Highly purified RNA was isolated from each hindgut and first strand cDNA was prepared with appropriate negative controls. Reverse transcriptase (RT) polymerase chain reaction (PCR) was done to determine the transcripts of Shh in each sample and quantitative real-time PCR was carried out to show relative quantitative expression of Shh at each time point. Shh was detected in all samples confirming that Shh is active during the process of hindgut development in fetal rats. Relative quantitation demonstrated that Shh expression shows time-dependent changes in the developing hindgut of ETU-exposed rat embryos, and when results were compared with control samples, there was significant decrease in expression on gD14 and 15, when the cloaca normally separates into the rectum and urethra occurs in the rat fetus. The misregulated expression of Shh in the hindgut of ETU-exposed rat embryos suggests that ETU may interfere with Shh signalling. Downregulation at the time of cloacal separation into rectum and urethra indicates that Shh plays a crucial role in the development of hindgut.

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References

  1. Nusslein-Volhard C, Wieschaus E (1980) Mutations affecting segment number and polarity in Drosophila. Nature 287(5785):795–801

    Article  PubMed  CAS  Google Scholar 

  2. Hammerschmidt M, Brook A, McMahon AP (1997) The world according to hedgehog. Trends Genet 13(1):14–21

    Article  PubMed  CAS  Google Scholar 

  3. Echelard Y, Epstein DJ, St-Jacques B et al (1993) Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75(7):1417–1430

    Article  PubMed  CAS  Google Scholar 

  4. Ekker SC, Ungar AR, Greenstein P et al (1995) Patterning activities of vertebrate hedgehog proteins in the developing eye and brain. Curr Biol 5(8):944–955

    Article  PubMed  CAS  Google Scholar 

  5. Krauss S, Concordet JP, Ingham PW (1993) A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75(7):1431–1444

    Article  PubMed  CAS  Google Scholar 

  6. Bronner-Fraser M, Fraser SE (1997) Differentiation of the vertebrate neural tube. Curr Opin Cell Biol 9(6):885–891

    Article  PubMed  CAS  Google Scholar 

  7. Bumcrot DA, McMahon AP (1995) Somite differentiation. Sonic signals somites. Curr Biol 5(6):612–614

    Article  PubMed  CAS  Google Scholar 

  8. Riddle RD, Johnson RL, Laufer E et al (1993) Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75(7):1401–1416

    Article  PubMed  CAS  Google Scholar 

  9. Roberts DJ, Johnson RL, Burke AC et al (1995) Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut. Development 121(10):3163–3174

    PubMed  CAS  Google Scholar 

  10. Ingham PW, McMahon AP (2001) Hedgehog signaling in animal development: paradigms and principles. Genes Dev 15(23):3059–3087

    Article  PubMed  CAS  Google Scholar 

  11. Marigo V, Davey RA, Zuo Y et al (1996) Biochemical evidence that patched is the Hedgehog receptor. Nature 384(6605):176–179

    Article  PubMed  CAS  Google Scholar 

  12. Alcedo J, Ayzenzon M, Von Ohlen T et al (1996) The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal. Cell 86(2):221–232

    Article  PubMed  CAS  Google Scholar 

  13. Ramalho-Santos M, Melton DA, McMahon AP (2000) Hedgehog signals regulate multiple aspects of gastrointestinal development. Development 127(12):2763–2772

    PubMed  CAS  Google Scholar 

  14. Sukegawa A, Narita T, Kameda T et al (2000) The concentric structure of the developing gut is regulated by Sonic hedgehog derived from endodermal epithelium. Development 127(9):1971–1980

    PubMed  CAS  Google Scholar 

  15. Kiel EM, Pena A (1998) Anorectal malformations. In: O’Nell J, Rowe MI, Grosfeld JL (eds) Paediatric surgery. Mosby, St. Louis, pp 1425–1448

    Google Scholar 

  16. Bitgood MJ, McMahon AP (1995) Hedgehog and Bmp genes are coexpressed at many diverse sites of cell–cell interaction in the mouse embryo. Dev Biol 172(1):126–138

    Article  PubMed  CAS  Google Scholar 

  17. Roberts DJ (1999) Embryology of the gastrointestinal tract. In: Sanderson IR, Walker WA (eds) Development of the gastrointestinal tract. B.C. Decker, Hamilton, pp 1–12

    Google Scholar 

  18. Wells JM, Melton DA (2000) Early mouse endoderm is patterned by soluble factors from adjacent germ layers. Development 127(8):1563–1572

    PubMed  CAS  Google Scholar 

  19. Kim J, Kim P, Hui CC (2001) The VACTERL association: lessons from the Sonic hedgehog pathway. Clin Genet 59(5):306–315

    Article  PubMed  CAS  Google Scholar 

  20. Kim PC, Mo R, Hui Cc C (2001) Murine models of VACTERL syndrome: role of sonic hedgehog signaling pathway. J Pediatr Surg 36(2):381–384

    Article  PubMed  CAS  Google Scholar 

  21. Kimmel SG, Mo R, Hui CC et al (2000) New mouse models of congenital anorectal malformations (discussion 230–221). J Pediatr Surg 35(2):227–230

    Article  PubMed  CAS  Google Scholar 

  22. Kondo T, Dolle P, Zakany J et al (1996) Function of posterior HoxD genes in the morphogenesis of the anal sphincter. Development 122(9):2651–2659

    PubMed  CAS  Google Scholar 

  23. Mo R, Kim JH, Zhang J et al (2001) Anorectal malformations caused by defects in sonic hedgehog signaling. Am J Pathol 159(2):765–774

    PubMed  CAS  Google Scholar 

  24. Hirai Y, Kuwabara N (1990) Transplacentally induced anorectal malformations in rats. J Pediatr Surg 25(7):812–816

    Article  PubMed  CAS  Google Scholar 

  25. Qi BQ, Beasley SW, Frizelle FA (2002) Clarification of the processes that lead to anorectal malformations in the ETU-induced rat model of imperforate anus. J Pediatr Surg 37(9):1305–1312

    Article  PubMed  Google Scholar 

  26. Vandesompele J, De Preter K, Pattyn F et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):RESEARCH0034

    Google Scholar 

  27. Diez-Pardo JA, Marino JM, Baoquan Q et al (1995) Neural tube defects: an experimental model in the foetal rat. Eur J Pediatr Surg 5(4):198–202

    Article  PubMed  CAS  Google Scholar 

  28. Roberts DJ, Smith DM, Goff DJ et al (1998) Epithelial–mesenchymal signaling during the regionalization of the chick gut. Development 125(15):2791–2801

    PubMed  CAS  Google Scholar 

  29. Tourneax F (1888) Sur le premiers developments due to cloaque du tubercle genitale et de l’anus chez l’embryon moutons, avec quelques remarques concernant le development des grlandes prostatiques. J Aant Physio 24:503–517

    Google Scholar 

  30. Retterer E (1890) Sur l’origin et de l’evolution de la r’egion ano-genitale des mammiferes. J Aant Physio 26:126–210

    Google Scholar 

  31. Kluth D, Hillen M, Lambercht W (1995) The principles of normal and abnormal hindgut development. J Pediatr Surg 30:1143–1147

    Article  PubMed  CAS  Google Scholar 

  32. van der Putte SC (1986) Normal and abnormal development of the anorectum. J Pediatr Surg 21:434–440

    Article  PubMed  Google Scholar 

  33. Qi BQ, Beasley SW, Arsic D (2004) Abnormalities of the vertebral column and ribs associated with anorectal malformations. Pediatr Surg Int 20(7):529–533

    Article  PubMed  Google Scholar 

  34. Qi BQ, Beasley SW, Frizelle FA (2003) Evidence that the notochord may be pivotal in the development of sacral and anorectal malformations. J Pediatr Surg 38(9):1310–1316

    Article  PubMed  Google Scholar 

  35. Qi BQ, Williams A, Beasley S et al (2000) Clarification of the process of separation of the cloaca into rectum and urogenital sinus in the rat embryo. J Pediatr Surg 35(12):1810–1816

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We wish to thank Dr. Glen Reid, Salim Ismail, Dr. Jacqueline Keenan (Department of Surgery), and Dr. Leigh Ellmers (Cardioendocrine Research Group) for their constructive suggestions and excellent technical assistance.

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Authors and Affiliations

  1. Children’s Cancer and Developmental Research Group, Department of Paediatric and Surgery, Christchurch School of Medicine & Health Sciences, Private Bag 4710, Christchurch, New Zealand

    Parkash Mandhan, Spencer Beasley, Tracy Hale & Michael Sullivan

  2. Cardioendocrine Research Group, Department of Medicine, Christchurch School of Medicine & Health Sciences, Christchurch, New Zealand

    Leigh Ellmers

  3. Department of Surgery, Christchurch School of Medicine & Health Sciences, Christchurch, New Zealand

    Justin Roake

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  1. Parkash Mandhan
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  2. Spencer Beasley
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  3. Tracy Hale
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  6. Michael Sullivan
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Correspondence to Michael Sullivan.

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Mandhan, P., Beasley, S., Hale, T. et al. Sonic hedgehog expression in the development of hindgut in ETU-exposed fetal rats. Ped Surgery Int 22, 31–36 (2006). https://doi.org/10.1007/s00383-005-1575-6

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  • DOI: https://doi.org/10.1007/s00383-005-1575-6

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