Hedgehog signaling displays a biphasic expression pattern during intestinal injury and repair Rui Liang a,b,d , Peter Morris b , Steven S.C. Cho b,d , Helen E. Abud d , Xianqing Jin a , Wei Cheng b,c, ⁎ a Key Laboratory of Developmental Diseases in Childhood (Chongqing Medical University), Ministry of Education, China b Monash Institute of Medical Research, Monash University, Melbourne, Australia c Department of Paediatrics and Department of Surgery, Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne, Australia d Department of Anatomy & Developmental Biology, Monash University, Melbourne, Australia Received 30 August 2012; accepted 1 September 2012 Key words: Hedgehog signaling; Gastrointestinal injury; Small intestinal epithelium Abstract Background/Purpose: Gastrointestinal injury is common clinically. The exact mechanism by which gastrointestinal repair occurs has yet to be well defined. Hedgehog (Hh) signaling is known to be involved in gastrointestinal development and repair of tissues such as skin and heart. The present study aimed to investigate the role of Hh in the repair of the small intestine. Methods: i) To study acute intestinal injury, we optimized a mouse model of 5-flurouracil (5-FU) induced injury of the small intestine. Ileal tissues were evaluated for injury and repair markers at day 0, 2, 5, and 9. ii) Immunohistochemistry (Sonic hedgehog, Shh), in situ hybridization (Shh), and Ptch/LacZ transgenic mice were carried out to localize hedgehog expression. A33CrPr × ShhTg knock-in mice were bred to study the effect of Shh over-expression. qPCR of Shh, Ihh, Ptch, Bmp4 was carried out to quantify hedgehog signaling. iii) 5FU treated mice were then treated with a hedgehog inhibitor or saline (control) and the effects of Shh inhibition including apoptosis, proliferation, and mitosis were then compared. Results: i) Immunohistochemistry and in situ hybridization of Shh, qPCR of hedgehog signaling pathway genes, and Ptch/LacZ staining results consistently showed down-regulation during the injury phase (P b 0.05) followed by up-regulation during the repair phase (P b 0.005). ii) Hh signaling inhibition following 5-FU induced injury augmented apoptotic activity (P b 0.05), suppressed mitotic activity (P b 0.005) in intestinal crypts, and reduced Paneth cell hyperplasia (P b 0.005). iii) Shh over-expression in conditionally knock-mice led to increased mitotic, Paneth, and goblet cells. Conclusion: Hedgehog signaling pathway displays a biphasic expression pattern during the injury/repair of small intestine. It may play an important regulatory role in intestinal repair. © 2012 Elsevier Inc. All rights reserved. Intestinal injury results from various clinical conditions. This includes ischaemia–reperfusion injury, inflammatory bowel disease and gastroenteritis as well as enterocolitis after cytotoxic chemotherapy. The intestinal epithelium does, ⁎ Corresponding author. Clayton, Victoria 3168, Australia. Tel.: +61 03 9594 5674; fax: +61 03 9594 6495. E-mail address: wei.cheng@monash.edu (W. Cheng). www.elsevier.com/locate/jpedsurg 0022-3468/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpedsurg.2012.09.016 Journal of Pediatric Surgery (2012) 47, 2251–2263