The zebraﬁsh mutant lessen: an experimental model for congenital enteric neuropathies L. UYTTEBROEK,* I. T. SHEPHERD, † P. VANDEN BERGHE, ‡ G. HUBENS,* J.-P. TIMMERMANS§ & L. VAN NASSAUW* *Laboratory of Human Anatomy and Embryology, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerpen, Belgium †Department of Biology, Emory University, Atlanta, GA, USA ‡Laboratory for Enteric NeuroScience (LENS), Translational Research in GastroIntestinal Disorders (TARGID), Department of Clinical and Experimental Medicine, University of Leuven, Leuven, Belgium §Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerpen, Belgium Key Messages • The aim of the study was to characterize the enteric phenotype of the zebraﬁsh mutant lessen. • Wild-type and lessen zebraﬁsh were used in vivo at 3–6 days post fertilization to investigate the development of intestinal motility patterns, while the intrinsic innervation and the ICC network were investigated in the intestinal segments using immunohistochemistry. • Mutant zebraﬁsh show a disturbed and delayed onset of intestinal motility, accompanied by a disturbed formation of the ENS and ICC network. • The distal parts of the intestine were most affected in the lessen mutant, while the proximal and mid parts were less affected. • The zebraﬁsh mutant lessen shows a defect in the development of the ENS, ICC network and intestinal motility, and may serve as an appropriate animal model to investigate the pathogenesis of CEN. Abstract Background Congenital enteric neuropathies of the distal intestine (CEN) are characterized by the partial or complete absence of enteric neurons. Over the last decade, zebraﬁsh has emerged as a leading model organism in experimental research. Our aim was to demonstrate that the mutant zebraﬁsh, lessen, expressing CEN characteristics, is an equally valu- able animal model alongside mammalian models for CEN, by studying its enteric phenotype. Methods The effect of the lessen mutation on the development of the enteric nervous system (ENS), interstitial cells of Cajal (ICC), and intestinal motility in each intestinal region of mutant and wild-type (wt) zebraﬁsh embryos at 3–6 dpf, was analyzed by immunoﬂuo- rescent detection of neurochemical markers and motility assays. Key Results Development of intesti- nal motility in the mutant was delayed and the majority of the observed contractions were disturbed. A signiﬁcant disturbance in ENS development resulted in a distal intestine that was almost free of neuronal elements, in reduced neuronal density in the proximal and mid-intestine, and in a defect in the expression of neurochemical markers. Furthermore, markedly disturbed development of ICC gave rise to a less dense network of ICC. Conclusions & Infer- ences The observed alterations in intestinal motility, intrinsic innervation and ICC network of the mutant in comparison with the wt zebraﬁsh, are similar to those seen in the oligo- and aganglionic regions of the intestine of CEN patients. It is concluded that the zebraﬁsh mutant lessen is an appropriate animal model to investigate CEN. Address for Correspondence Dr. Leen Uyttebroek, Laboratory of Human Anatomy and Embryology, Faculty of Medicine and Health Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerpen, Belgium. Tel: +32 32 65 28 28; e-mail: leen.uyttebroek@uantwerpen.be Received: 27 March 2015 Accepted for publication: 22 October 2015 © 2015 John Wiley & Sons Ltd 345 Neurogastroenterol Motil (2016) 28, 345–357 doi: 10.1111/nmo.12732 Neurogastroenterology & Motility