Embryonic Wnt gene expression in the nitrofen-induced hypoplastic lung using 3-dimensional imaging Hajime Takayasu a , Paula Murphy b , Hideaki Sato a , Takashi Doi a,c , Prem Puri a,c, ⁎ a The Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland b Department of Zoology, School of Natural Sciences, Trinity College, Dublin, Ireland c School of Medicine and Medical Science and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland Received 22 January 2010; revised 24 June 2010; accepted 28 June 2010 Key words: Congenital diaphragmatic hernia; Optical projection tomography; Whole-mount in situ hybridization; Wnt; Nitrofen Abstract Purpose: Wnts have been reported to play a key role in the lung morphogenesis. We have previously reported that pulmonary gene expression of Wnt2 and Wnt7b is downregulated on day 15 of gestation in the nitrofen-induced congenital diaphragmatic hernia (CDH) model. However, the distribution pattern of gene expression of Wnts in the very early lung development remains unclear. Optical projection tomography (OPT) is a new technique for 3-dimensional imaging of small developing organs and gene distribution combined with whole-mount in situ hybridization. We designed this study to investigate the distribution pattern of Wnts gene expression in lung buds of nitrofen-induced CDH model using OPT. Methods: Embryos from normal and nitrofen-treated dams were harvested on embryonic day 10 (E10), and divided into controls and nitrofen group, respectively. Whole-mount in situ hybridization to detect transcripts of Wnt2 and Wnt7b was performed, analyzed, and reconstructed using OPT. Results: The expression of Wnt2 transcripts was detected in the lung bud mesenchyme and markedly diminished in nitrofen group compared to controls, whereas Wnt7b transcripts were expressed in the mesoderm of bronchi and the lung bud with no detectable difference between 2 groups. Conclusion: We provide evidence for the first time that Wnt2 expression is downregulated at lung bud stage in the nitrofen model. Optical projection tomography is potentially a useful approach to visualize both gene expression and morphology during very early stages of lung development. © 2010 Elsevier Inc. All rights reserved. Despite improved understanding of the pathophysiology of congenital diaphragmatic hernia (CDH) and advances in perinatal care, the prognosis of infants with severe CDH remains poor [1,2]. Hypoplastic lung and persistent pulmo- nary hypertension are the principle causes of the high morbidity and mortality in infants with CDH [1-3]. The affected infants show developmental retardation of lung, including fewer bronchial branches and alveoli, and retarda- tion of alveolar development [4-6]. The pathogenesis of pulmonary hypoplasia associated with CDH is not fully understood. In the nitrofen-induced CDH animal model, some investigators reported that nonmechanical factors directory mediated by nitrofen play a significant role in the ⁎ Corresponding author. The Children's Research Centre, Our Lady's Children's Hospital, Dublin 12, Ireland. Tel.: +353 1 4096420; fax: +353 1 4550201. E-mail address: prem.puri@ucd.ie (P. Puri). www.elsevier.com/locate/jpedsurg 0022-3468/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2010.06.046 Journal of Pediatric Surgery (2010) 45, 2129–2135